Diana M. Mate

Laboratory Evolution of High-Redox Potential Laccases

Thermostable laccases with a high-redox potential have been engineered through a strategy that combines directed evolution with rational approaches. The original laccase signal sequence was replaced by the α-factor prepro-leader, and the corresponding fusion gene was targeted for joint laboratory evolution with the aim of improving kinetics and secretion by Saccharomyces cerevisiae, while retaining high thermostability. After eight rounds of molecular evolution, the total laccase activity was enhanced 34,000-fold culminating in the OB-1 mutant as the last variant of the evolution process, a highly active and stable enzyme in terms of temperature, pH range, and organic cosolvents. Mutations in the hydrophobic core of the evolved α-factor prepro-leader enhanced functional expression, whereas some mutations in the mature protein improved its catalytic capacities by altering the interactions with the surrounding residues.

Self-powered wireless carbohydrate/oxygen sensitive biodevice based on radio signal transmission

Here for the first time, we detail self-contained (wireless and self-powered) biodevices with wireless signal transmission. Specifically, we demonstrate the operation of self-sustained carbohydrate and oxygen sensitive biodevices, consisting of a wireless electronic unit, radio transmitter and separate sensing bioelectrodes, supplied with electrical energy from a combined multi-enzyme fuel cell generating sufficient current at required voltage to power the electronics. A carbohydrate/oxygen enzymatic fuel cell was assembled by comparing the performance of a range of different bioelectrodes followed by selection of the most suitable, stable combination. Carbohydrates (viz. lactose for the demonstration) and oxygen were also chosen as bioanalytes, being important biomarkers, to demonstrate the operation of the self-contained biosensing device, employing enzyme-modified bioelectrodes to enable the actual sensing. A wireless electronic unit, consisting of a micropotentiostat, an energy harvesting module (voltage amplifier together with a capacitor), and a radio microchip, were designed to enable the biofuel cell to be used as a power supply for managing the sensing devices and for wireless data transmission. The electronic system used required current and voltages greater than 44 µA and 0.57 V, respectively to operate; which the biofuel cell was capable of providing, when placed in a carbohydrate and oxygen containing buffer. In addition, a USB based receiver and computer software were employed for proof-of concept tests of the developed biodevices. Operation of bench-top prototypes was demonstrated in buffers containing different concentrations of the analytes, showcasing that the variation in response of both carbohydrate and oxygen biosensors could be monitored wirelessly in real-time as analyte concentrations in buffers were changed, using only an enzymatic fuel cell as a power supply.

Evolving thermostability in mutant libraries of ligninolytic oxidoreductases expressed in yeast

BackgroundIn the picture of a laboratory evolution experiment, to improve the thermostability whilst maintaining the activity requires of suitable procedures to generate diversity in combination with robust high-throughput protocols. The current work describes how to achieve this goal by engineering ligninolytic oxidoreductases (a high-redox potential laccase -HRPL- and a versatile peroxidase, -VP-) functionally expressed in Saccharomyces cerevisiae.ResultsTaking advantage of the eukaryotic machinery, complex mutant libraries were constructed by different in vivo recombination approaches and explored for improved stabilities and activities. A reliable high-throughput assay based on the analysis of T50 was employed for discovering thermostable oxidases from mutant libraries in yeast. Both VP and HRPL libraries contained variants with shifts in the T50 values. Stabilizing mutations were found at the surface of the protein establishing new interactions with the surrounding residues.ConclusionsThe existing tradeoff between activity and stability determined from many point mutations discovered by directed evolution and other protein engineering means can be circumvented combining different tools of in vitro evolution.

Laccase: a multi‐purpose biocatalyst at the forefront of biotechnology

Laccases are multicopper containing enzymes capable of performing one electron oxidation of a broad range of substrates. Using molecular oxygen as the final electron acceptor, they release only water as a by‐product, and as such, laccases are eco‐friendly, versatile biocatalysts that have generated an enormous biotechnological interest. Indeed, this group of enzymes has been used in different industrial fields for very diverse purposes, from food additive and beverage processing to biomedical diagnosis, and as cross‐linking agents for furniture construction or in the production of biofuels. Laccases have also been studied intensely in nanobiotechnology for the development of implantable biosensors and biofuel cells. Moreover, their capacity to transform complex xenobiotics makes them useful biocatalysts in enzymatic bioremediation. This review summarizes the most significant recent advances in the use of laccases and their future perspectives in biotechnology.

Development of Chimeric Laccases by Directed Evolution

DNA recombination methods are useful tools to generate diversity in directed evolution protein engineering studies. We have designed an array of chimeric laccases with high‐redox potential by in vitro and in vivo DNA recombination of two fungal laccases (from Pycnoporus cinnabarinus and PM1 basidiomycete), which were previously tailored by laboratory evolution for functional expression in Saccharomyces cerevisiae. The laccase fusion genes (including the evolved α‐factor prepro‐leaders for secretion in yeast) were subjected to a round of family shuffling to construct chimeric libraries and the best laccase hybrids were identified in dual high‐throughput screening (HTS) assays. Using this approach, we identified chimeras with up to six crossover events in the whole sequence, and we obtained active hybrid laccases with combined characteristics in terms of pH activity and thermostability. Biotechnol. Bioeng. 2012; 109: 2978–2986.

Directed evolution of fungal laccases.

Fungal laccases are generalists biocatalysts with potential applications that range from bioremediation to novel green processes. Fuelled by molecular oxygen, these enzymes can act on dozens of molecules of different chemical nature, and with the help of redox mediators, their spectrum of oxidizable substrates is further pushed towards xenobiotic compounds (pesticides, industrial dyes, PAHs), biopolymers (lignin, starch, cellulose) and other complex molecules. In recent years, extraordinary efforts have been made to engineer fungal laccases by directed evolution and semi-rational approaches to improve their functional expression or stability. All these studies have taken advantage of Saccharomyces cerevisiae as a heterologous host, not only to secrete the enzyme but also, to emulate the introduction of genetic diversity through in vivo DNA recombination. Here, we discuss all these endeavours to convert fungal laccases into valuable biomolecular platforms on which new functions can be tailored by directed evolution.

Widening the pH Activity Profile of a Fungal Laccase by Directed Evolution

Unnatural selection: A fungal laccase was tailored by directed evolution to be active at neutral/alkaline pH. After five generations, the final mutant showed a broader pH profile while retaining 50 to 80 % of its activity at neutral pH.

Tandem-yeast expression system for engineering and producing unspecific peroxygenase

Unspecific peroxygenase (UPO) is a highly efficient biocatalyst with a peroxide dependent monooxygenase activity and many biotechnological applications, but the absence of suitable heterologous expression systems has precluded its use in different industrial settings. Recently, the UPO from Agrocybe aegerita was evolved for secretion and activity in Saccharomyces cerevisiae [8]. In the current work, we describe a tandem-yeast expression system for UPO engineering and large scale production. By harnessing the directed evolution process in S. cerevisiae, the beneficial mutations for secretion enabled Pichia pastoris to express the evolved UPO under the control of the methanol inducible alcohol oxidase 1 promoter. Whilst secretion levels were found similar for both yeasts in flask fermentation (∼8mg/L), the recombinant UPO from P. pastoris showed a 27-fold enhanced production in fed-batch fermentation (217mg/L). The P. pastoris UPO variant maintained similar biochemical properties of the S. cerevisiae counterpart in terms of catalytic constants, pH activity profiles and thermostability. Thus, this tandem-yeast expression system ensures the engineering of UPOs to use them in future industrial applications as well as large scale production.

Bioelectrochemical Oxidation of Water

The electrolysis of water provides a link between electrical energy and hydrogen, a high energy density fuel and a versatile energy carrier, but the process is very expensive. Indeed, the main challenge is to reduce energy consumption for large-scale applications using efficient renewable catalysts that can be produced at low cost. Here we present for the first time that laccase can catalyze electrooxidation of H2O to molecular oxygen. Native and laboratory-evolved laccases immobilized onto electrodes serve as bioelectrocatalytic systems with low overpotential and a high O2 evolution ratio against H2O2 production during H2O electrolysis. Our results open new research ground on H2O splitting, as they overcome serious practical limitations associated with artificial electrocatalysts currently used for O2 evolution.

Functional expression of a blood tolerant laccase in Pichia pastoris.

BackgroundBasidiomycete high-redox potential laccases (HRPLs) working in human physiological fluids (pH 7.4, 150 mM NaCl) arise great interest in the engineering of 3D-nanobiodevices for biomedical uses. In two previous reports, we described the directed evolution of a HRPL from basidiomycete PM1 strain CECT 2971: i) to be expressed in an active, soluble and stable form in Saccharomyces cerevisiae, and ii) to be active in human blood. In spite of the fact that S. cerevisiae is suited for the directed evolution of HRPLs, the secretion levels obtained in this host are not high enough for further research and exploitation. Thus, the search for an alternative host to over-express the evolved laccases is mandatory.ResultsA blood-active laccase (ChU-B mutant) fused to the native/evolved α-factor prepro-leader was cloned under the control of two different promoters (PAOX1 and PGAP) and expressed in Pichia pastoris. The most active construct, which contained the PAOX1 and the evolved prepro-leader, was fermented in a 42-L fed-batch bioreactor yielding production levels of 43 mg/L. The recombinant laccase was purified to homogeneity and thoroughly characterized. As happened in S. cerevisiae, the laccase produced by P. pastoris presented an extra N-terminal extension (ETEAEF) generated by an alternative processing of the α-factor pro-leader at the Golgi compartment. The laccase mutant secreted by P. pastoris showed the same improved properties acquired after several cycles of directed evolution in S. cerevisiae for blood-tolerance: a characteristic pH-activity profile shifted to the neutral-basic range and a greatly increased resistance against inhibition by halides. Slight biochemical differences between both expression systems were found in glycosylation, thermostability and turnover numbers.ConclusionsThe tandem-yeast system based on S. cerevisiae to perform directed evolution and P. pastoris to over-express the evolved laccases constitutes a promising approach for the in vitro evolution and production of these enzymes towards different biocatalytic and bioelectrochemical applications.