Application of hydrocarbon and perfluorocarbon oxygen vectors to enhance heterologous production of hyaluronic acid in engineered Bacillus subtilis

Abstract

In microbial cultivations for hyaluronic acid (HA) production, oxygen can be a limiting substrate due to its poor solubility in aqueous medium and the substantial increase in culture viscosity at relatively low HA titers. Shear stress due to the high agitation and aeration rates required to overcome oxygen limitation may reduce the quality (i.e., molecular weight) of HA, and production costs associated with power consumption and supplemental oxygen may be excessive. Here, we report the application of oxygen vectors to the heterologous production of HA in engineered Bacillus subtilis, leading to significantly improved culture performance. We first derived an improved HA-producing strain of B. subtilis through engineering of the promoter driving coexpression of seHas and tuaD, leading to high-level HA production. Out of seven potential oxygen vectors evaluated in a preliminary screening, significant improvements to the HA titer and/or cell density were observed in cultures containing n-heptane, n-hexadecane, perfluoromethyldecalin, and perfluoro-1,3-dimethylcyclohexane. Adjustments to the vector concentration, timing of vector addition, and the agitation rate resulted in further enhancements, with the HA titer reaching up to 4.5 g/L after only 10 hr cultivation. Moreover, our results indicate that certain vectors may alter the functional expression of Class I hyaluronan synthase (HAS) in B. subtilis, and that higher shear rates may drive more carbon flux through the HA biosynthetic pathway without negatively affecting the MW. Our study demonstrates the efficacy of oxygen vectors to enhance heterologous HA production in B. subtilis, and provides valuable insight for future bioprocess development in microbial HA production.

Genetic strategies for improving hyaluronic acid production in recombinant bacterial culture

“…Nevertheless, the latter strategy appears to depend on which organism is utilized: in C. glutamicum, unlike in B. subtilis, the expression of vhb had a negative impact on HA synthesis (Hoffmann & Altenbuchner, 2014). Recently, it was reported that utilization of oxygen carriers increases HA titre in a B. subtilis HA-producing strain (Westbrook et al, 2018c). These are hydrophobic compounds with a higher capacity to dissolve oxygen than water, enhancing oxygen transfer in microbial cultures.…”

“…Recently, it was reported that utilization of oxygen carriers increases HA titre in a B . subtilis HA‐producing strain (Westbrook et al, 2018c). These are hydrophobic compounds with a higher capacity to dissolve oxygen than water, enhancing oxygen transfer in microbial cultures.…”

Enhancement of interfacial catalysis in a triphase reactor using oxygen nanocarriers

“…Many liquids, such as perfluorocarbon and silicone oils, are biologically inert and have oxygen solubility up to one order of magnitude higher than that of water [20,21]. Taking advantage of these characteristics, perfluorocarbon has been used as a blood substitute [22][23][24] and in microbial reactors for enhanced biofuel production [25]. However, efficient biocatalytic reactions that use such a second liquid phase are still difficult to conduct, because the interaction efficiency between water and oil immiscible phases is extremely low.…”

Amphiphilic Fluorine-Containing Block Copolymers as Carriers for Hydrophobic PtTFPP for Dissolved Oxygen Sensing, Cell Respiration Monitoring and In Vivo Hypoxia Imaging with High Quantum Efficiency and Long Lifetime

“…It has been reported that micelles formed from fluorine-containing amphiphilic polymers exhibited high stability and nontoxicity which can be used as protein drug carriers [ 30 ]. Based on the former research on perfluorocarbon oxyglobin, fluorine has greater capacity to dissolve, carry and permeate oxygen [ 31 ]. Besides, the presence of halide substituents could increase the photostability of metalloporphyrins [ 32 ].…”