Production of Chloroplast-Derived Endoglucanases

Plant chloroplast genetic engineering can utilize transgenic crops to express different enzymes from bacteria or fungi for the production of feedstocks for biofuels. Lifeasible has successfully developed a variety of reliable and economical solutions for enzymatic chloroplast engineering for biofuels. Our engineers focus on developing developing thermostable endoglucanases with heterologous expression for the biofuel industry to facilitate the biotransformation of cellulosic biomass.

Introduction

Endoglucanases are members of the cellulase family and have a high affinity for cellulose. Endoglucanases work by cleaving internal β-glycosidic bonds in cellulose chains. The final product, cellobiose, is further broken down into glucose units by beta-glucosidases, which are then fermented to produce biofuels. Endoglucanases obtained from fermentation of fungal and bacterial sources are often added to the production batch after a pretreatment step, adding to the final cost of the bioethanol product. Heterologous expression of endoglucanases in plants has long been proposed to improve biofuel production. With the development of biotechnology and genetic engineering, plants can achieve low-cost production of stable enzymes to produce industrially useful products. The first report on the production of endoglucanase in plants was in tobacco, in which the endoglucanase was targeted to the chloroplast.

Fig. 1. Cellulose nanocrystals were obtained by enzymatic hydrolysis using on-site produced enzymes and eucalyptus cellulose pulp in a sustainable integrated process. (Squinca P, et al., 2020)

Solutions

The high production cost of endoglucanases limits their commercial application. Transgenic plants via chloroplasts are ideal for cost-effective production of enzymes. We have successfully applied chloroplast transformation technology to the development of endoglucanases. Lifeasible is committed to the synthesis of endoglucanases using the EGph gene expressed in tobacco chloroplasts. Based on our knowledge of protein sequence, structure and dynamics, our engineers are designing thermostable endoglucanases and improving thermostability through the following strategies:

• Selective pressure for certain amino acids.
• Increase in hydrophobicity.
• Changes in a single amino acid.
• Increase in tightness.
• Positively charged amino acids increase.
• Gibbs free energy change of hydration.

Our goal is to provide an efficient and low-cost solution to develop feedstocks with heterologously expressed thermostable endoglucanases. Our customers have successfully used chloroplast-derived endoglucanases in the washing industry. The strategy of our solution is roughly as follows:

(1) The vector was constructed using a hyperthermostable β-1, 4endolucanase from the archaeon Pyrococcus horikoshii (EGph gene).
(2) Transform the vector into tobacco chloroplasts.
(3) PCR screening to detect the presence of EGph gene in tobacco chloroplast genome.
(4) Southern blot analysis of the integration, homogeneity and transcriptional stability of EGph protein.
(5) The thermostability of endoglucanase was analyzed.

Attractive Advantages of Our Solutions

• Introducing endoglucanases into plants reduces cellulose recalcitrance.
• Large-scale sustainable production of endoglucanases using plant systems.
• Industrial production of low-cost endoglucanase is realized without expensive equipment.
• Transgenic residues remaining after endoglucanase extraction can be used as feedstock from which fermentable sugars are produced.
• The expression of the hyperthermic endoglucanase gene was achieved.

Lifeasible is committed to providing customers around the world with fully customized chloroplast engineered solutions for endoglucanases production. Please contact us to discuss further details to ensure your next success.

References

1. Squinca P, Bilatto S, Badino A C, et al. (2020) Nanocellulose production in future biorefineries: An integrated approach using tailor-made enzymes[J]. ACS Sustainable Chemistry & Engineering. 8(5): 2277-2286.
2. Yennamalli R M, Rader A J, Kenny A J, et al. (2013) Endoglucanases: insights into thermostability for biofuel applications[J]. Biotechnology for biofuels. 6(1): 1-9.