The sustainable production of hydroxy fatty acids, such as 10-hydroxystearic acid (10-HSA), by biocatalysis is a promising alternative to petrochemical and castor oil-derived products. However, industrial implementation still requires an efficient and scalable process for biocatalyst production by fermentation, which precedes the biotransformation to 10-HSA. In general, industrial biocatalysis is commonly performed using whole cells, which are cheaper than purified enzymes. Still, the fermentation process itself is a major cost contributor requiring a high-yielding and scalable process. Here, we established and scaled a lactose-induced fed-batch process to produce an Escherichia coli BL21 (DE3) based whole-cell biocatalyst containing an oleate hydratase from Stenotrophomonas nitritireducens. The two-phased process employs an initial growth phase on glucose, followed by an induced feed phase using glycerol and lactose. When comparing two different growth rates during enzyme expression, a higher growth rate was found beneficial, resulting in a higher biomass concentration of 69.2 ± 0.5 g L-1 and a yield increase of 80 % while maintaining biocatalyst activity at >85 % conversion. This highlights the importance of process design variables, such as growth rate settings, for a high-yielding and economic process. Feasibility of the process was demonstrated by scaling into a 150 L bioreactor, achieving a biomass concentration of 60.6 ± 0.4 g L-1 with a yield of 0.44 gC,Biomass gC -1 and 91.5 ± 1.4 % conversion. Supplemented with an initial test of suitable unit operations for technical biomass separation, this work provides a fermentation route for a whole-cell oleate hydratase biocatalyst, paving the way for further scaling toward industrial 10-HSA production.
The biocatalytic conversion of green methanol, a promising feedstock, into high value-added products is seen as an attractive way to establish sustainable biomanufacturing. Although natural and engineered microorganisms can utilize methanol, efficiently converting C1 carbon into valuable chemicals in growing cells remains challenging because non-native pathways often suffer from low carbon utilization and limited catalytic stability. Here, we report the integration of an engineered enzyme cascade into resting cells and its coupling with a screened alcohol oxidase enzyme to develop an efficient and stable enzyme-resting cell cascade system for methanol conversion, achieving customizable production of L-erythrulose (C4 sugar) or L-sorbose (C6 sugar). This system shows a stimulated carbon atom economy for L-erythrulose production from methanol and significantly improved stability compared to free enzyme catalysis. By integrating enzyme cascades with durable resting cells, this strategy provides a versatile platform for converting methanol and other C1 substrates into value-added products. Methanol use in engineered methylotrophs is constrained by toxicity and low efficiency. Here, the authors report the engineering E. coli resting cells coupled with a screened alcohol oxidase to enable biocatalytic production of rare sugars from methanol as the sole substrate.
Natamycin is primarily produced via submerged fermentation (SmF), a process often limited by inefficient oxygen transfer, high agitation energy consumption, and the generation of substantial organic wastewater. This study introduces a semi-solid-state fermentation (SSSF) strategy using perlite carriers and corn straw hydrolysates (CSH) based substrates to enhance natamycin production. Under optimized conditions, 40% medium saturation, inoculation with 2.0 × 109 spores/L, 26 °C, and atomized nutrient supplementation, the SSSF process yielded 15.6 g/L of natamycin after 7 days, achieving a productivity of 2.2 g/L/day. The improvement is attributed to a more natural habitat that facilitates direct oxygen and nutrient transfer to the cells, accompanied by upregulated transcription of key biosynthetic genes, enhanced enzyme activity, improved energy metabolism, expanded pools of precursor metabolites, and strengthened antioxidative capacity. Owing to these advantages, SSSF reduced the total natamycin production cost by 83%, while eliminating fermentative wastewater and increasing natamycin productivity 2.9-fold versus glucose-based SmF. The SSSF strategy enables efficient natamycin biosynthesis with reduced energy input and no wastewater generation, demonstrating strong potential for industrial-scale application.
Lactobionic acid (LBA) is a compound that, in the last decade, has become critically important due to its potential applications in the food, chemical, pharmaceutical, and cosmetic industries. Enzymatic biosynthesis in the presence of a redox mediator is one method of producing LBA biologically. Cellobiose dehydrogenase (CDH) oxidizes the lactose to lactobionic acid, while laccase (LAC) enables the regeneration of the redox mediator (ABTS), which acts as an electron acceptor for CDH. The aim of this study was to develop an effective immobilized enzymatic system for the production of LBA. Two enzymes were used in the experiment: CDH from Phanerodontia chrysosporium (PchCDH) and LAC from Cerrena unicolor (CuLAC), which were immobilized on precipitated silica (Sipernat 22) activated by APTES and PEI. The immobilization process increased enzyme stability, improved the efficiency of LBA synthesis, and reduced costs, particularly in the context of using Sipernat 22 silica, which is inexpensive and widely used across various industries. The co-immobilization of both enzymes on the carrier proved to be the most effective approach, achieving a 90% conversion of lactose to lactobionic acid after ten cycles of synthesis. Comprehensive biochemical characterization, including protein loading, catalytic activity, and optimal pH, is provided in the main text.
Wiktoria Piątek-Gołda, Monika Osińska-Jaroszuk, M. Grąz et al.· Molecules· 0 citations
