Construction of engineered yeast producing ammonia from glutamine and soybean residues (okara)

Abstract

Ammonia is an essential substance for agriculture and the chemical industry. The intracellular production of ammonia in yeast (Saccharomyces cerevisiae) by metabolic engineering is difficult because yeast strongly assimilates ammonia, and the knockout of genes enabling this assimilation is lethal. Therefore, we attempted to produce ammonia outside the yeast cells by displaying a glutaminase (YbaS) from Escherichia coli on the yeast cell surface. YbaS-displaying yeast successfully produced 3.34 g/L ammonia from 32.6 g/L glutamine (83.2% conversion rate), providing it at a higher yield than in previous studies. Next, using YbaS-displaying yeast, we also succeeded in producing ammonia from glutamine in soybean residues (okara) produced as food waste from tofu production. Therefore, ammonia production outside cells by displaying ammonia-lyase on the cell surface is a promising strategy for producing ammonia from food waste as a novel energy resource, thereby preventing food loss.

Sustainable Biological Ammonia Production towards a Carbon-Free Society

“…Ammonia lyases are particularly suitable for display on the sur-face of yeast cells because they do not require cofactors for catalysis. In a study using yeast displaying glutamine ammonia-lyase YbaS, ammonia production from a glutamine solution succeeded with high efficiency (83.2%) and concentration (3.34 g/L) [104]. Additionally, although it is known that ammonia at concentrations higher than 0.1% (v/v) inhibited the growth of yeast, any impairment of the microbial catalyst was not observed due to ammonia toxicity in this approach [104,119].…”

“…Biomass, such as food waste, is also promising for producing various substances and as animal feed because it contains fixed nitrogen in various forms, such as proteins and nucleic acids [100][101][102][103][104][105][106]. For example, food waste can be used as organic fertiliser by digesting it with anaerobic bacteria [11].…”

“…Several attempts to produce ammonia from organic molecules containing nitrogen have been made (Table 1) [100,[102][103][104][105][106]. For example, there is a study using Bacillus subtilis [102].…”

“…Metabolic engineering Amino acids-based medium [102] Escherichia coli Metabolic engineering Amino acids-based medium [112] Escherichia coli Metabolic engineering Amino acids-based medium and soybean residue [106] Saccharomyces cerevisiae Cell surface engineering Glutamine solution and soybean residue [104] Saccharomyces cerevisiae Cell surface engineering Amino acids solution and soybean residue [105] Several attempts have been made to produce ammonia extracellularly. In some studies, yeast cell surface engineering systems have been used to avoid intracellular ammonia assimilation and toxicity [105,113,114].…”

“…Therefore, due to these advantages, yeast cell surface engineering has been successfully used to produce ethanol from carbohydrates, woody biomass, and macroalgae with high efficiency [116][117][118]. Furthermore, ammonia production from soybean residues has been attempted using yeast cell surface engineering [104]. Deaminase, transaminase, oxidase, and ammonia-lyase are known as amino acid catabolic enzymes that can produce ammonia from amino acids.…”