Molecular expression of a recombinant thermostable bacterial amylase from Geobacillus stearothermophilus SR74 using methanol-free Meyerozyma guilliermondii strain SO yeast system

α-Amylase, which was isolated from Geobacillus stearothermophilus SR74, has shown its potential to be used in industrial applications. However, its expression in the Pichia pastoris expression system with the alcohol oxidase 1 promoter (PAOX1) requires high methanol consumption and is time-consuming. This study aimed to express SR74 α-amylase in an alternative yeast system, using Meyerozyma guilliermondii strain SO, which was isolated from a spoiled orange (SO) under the regulation of a formaldehyde dehydrogenase promoter (PFLD). Qualitative screening showed that strain SO possessed a native amylase grown on YPD-starch plate at 30 °C. The recombinant SR74 α-amylase was further quantified and validated using the Western blot test. It was confirmed that SR74 α-amylase was expressed by strain SO extracellularly with a size of 59 kDa. Optimization in a shake flask showed that the recombinant SR74 α-amylase, which was regulated by PFLD, was successfully produced (26 U/mL) without any external inducer in the YPT medium after 24 h of cultivation. In conclusion, strain SO was able to produce SR74 amylase without methanol in one-fifth the fermentation time of P. pastoris. Further optimization of the expression may be done to improve the yield, as this methanol-free host is still underexplored.

Toward engineeringE. coliwith an autoregulatory system for lignin valorization

Efficient lignin valorization could add more than 10-fold the value gained from burning it for energy and is critical for economic viability of future biorefineries. However, lignin-derived aromatics from biomass pretreatment are known to be potent fermentation inhibitors in microbial production of fuels and other value-added chemicals. In addition, isopropyl-β-d-1-thiogalactopyranoside and other inducers are routinely added into fermentation broth to induce the expression of pathway enzymes, which further adds to the overall process cost. An autoregulatory system that can diminish the aromatics’ toxicity as well as be substrate-inducible can be the key for successful integration of lignin valorization into future lignocellulosic biorefineries. Toward that goal, in this study an autoregulatory system is demonstrated that alleviates the toxicity issue and eliminates the cost of an external inducer. Specifically, this system is composed of a catechol biosynthesis pathway coexpressed with an active aromatic transporter CouP under induction by a vanillin self-inducible promoter, ADH7, to effectively convert the lignin-derived aromatics into value-added chemicals usingEscherichia colias a host. The constructed autoregulatory system can efficiently transport vanillin across the cell membrane and convert it to catechol. Compared with the system without CouP expression, the expression of catechol biosynthesis pathway with transporter CouP significantly improved the catechol yields about 30% and 40% under promoter pTrc and ADH7, respectively. This study demonstrated an aromatic-induced autoregulatory system that enabled conversion of lignin-derived aromatics into catechol without the addition of any costly, external inducers, providing a promising and economically viable route for lignin valorization.