Chie Imamura

Advanced evolutionary molecular engineering to produce thermostable cellulase by using a small but efficient library.

We aimed at constructing thermostable cellulase variants of cellobiohydrolase II, derived from the mesophilic fungus Phanerochaete chrysosporium, by using an advanced evolutionary molecular engineering method. By aligning the amino acid sequences of the catalytic domains of five thermophilic fungal CBH2 and PcCBH2 proteins, we identified 45 positions where the PcCBH2 genes differ from the consensus sequence of two to five thermophilic fungal CBH2s. PcCBH2 variants with the consensus mutations were obtained by a cell-free translation system that was chosen for easy evaluation of thermostability. From the small library of consensus mutations, advantageous mutations for improving thermostability were found to occur with much higher frequency relative to a random library. To further improve thermostability, advantageous mutations were accumulated within the wild-type gene. Finally, we obtained the most thermostable variant Mall4, which contained all 15 advantageous mutations found in this study. This variant had the same specific cellulase activity as the wild type and retained sufficient activity at 50°C for >72 h, whereas wild-type PcCBH2 retained much less activity under the same conditions. The history of the accumulation process indicated that evolution of PcCBH2 toward improved thermostability was ideally and rapidly accomplished through the evolutionary process employed in this study.

Characterization of five terminator regions that increase the protein yield of a transgene in Saccharomyces cerevisiae.

Strong terminator regions could be used to improve metabolically engineered yeasts by increasing the target enzyme protein yields above those achieved with traditional terminator regions. We recently identified five strong terminator regions (RPL41Bt, RPL15At, DIT1t, RPL3t, and IDP1t) in a comprehensive analysis of Saccharomyces cerevisiae. The effect of the terminator regions was analyzed by measuring the protein production of a linked transgene, and was shown to be twice that of a traditional terminator region (PGK1t). Here, we investigated whether the activity of the terminator regions is affected by exchange of a strong promoter or reporter in the linked transgene, carbon source for cell growth, stress factors, host yeast strain, or stage of the growth phase. Our results indicate that the activities of all five terminator regions were twice that of PGK1t in all conditions tested. In addition, we demonstrated that the strong activity of these terminator regions could be used to improve secretory production of endoglucanase II derived from Tricoderma ressei, and that the DIT1t strain was the best of the five strains for this purpose. We therefore propose that DIT1t, and the four other terminator regions, could be applied to the development of improved metabolically engineered yeasts.

Overexpression of a novel Arabidopsis PP2C isoform, AtPP2CF1, enhances plant biomass production by increasing inflorescence stem growth

In contrast to mammals, higher plants have evolved to express diverse protein phosphatase 2Cs (PP2Cs). Of all Arabidopsis thaliana PP2Cs, members of PP2C subfamily A, including ABI1, have been shown to be key negative regulators of abscisic acid (ABA) signalling pathways, which regulate plant growth and development as well as tolerance to adverse environmental conditions. However, little is known about the enzymatic and signalling roles of other PP2C subfamilies. Here, we report a novel Arabidopsis subfamily E PP2C gene, At3g05640, designated AtPP2CF1. AtPP2CF1 was dramatically expressed in response to exogenous ABA and was expressed in vascular tissues and guard cells, similar to most subfamily A PP2C genes. In vitro enzymatic activity assays showed that AtPP2CF1 possessed functional PP2C activity. However, yeast two-hybrid analysis revealed that AtPP2CF1 did not interact with PYR/PYL/RCAR receptors or three SnRK2 kinases, which are ABI1-interacting proteins. This was supported by homology-based structural modelling demonstrating that the putative active- and substrate-binding site of AtPP2CF1 differed from that of ABI1. Furthermore, while overexpression of ABI1 in plants induced an ABA-insensitive phenotype, Arabidopsis plants overexpressing AtPP2CF1 (AtPP2CF1oe) were weakly hypersensitive to ABA during seed germination and drought stress. Unexpectedly, AtPP2CF1oe plants also exhibited increased biomass yield, mainly due to accelerated growth of inflorescence stems through the activation of cell proliferation and expansion. Our results provide new insights into the physiological significance of AtPP2CF1 as a candidate gene for plant growth production and for potential application in the sustainable supply of plant biomass.

Enhancement of Thermal Stabilization of Formaldehyde Dehydrogenase from Pseudomonas putida by Directed Evolution

We used directed evolution to enhance the thermostability of formaldehyde dehydrogenase from Pseudomonas putida. At 50 °C, the wild-type enzyme was inactivated within 30 min, but the variants obtained retained 80% activity for at least 300 min. At room temperature (30 °C), the variants obtained retained <80% activity for at least 500 h (21 d).

Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System

Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering.