Metabolic Engineering of Bacillus amyloliquefaciens to Efficiently Synthesize L-Ornithine From Inulin

Abstract

Bacillus amyloliquefaciens is the dominant strain used to produce γ-polyglutamic acid from inulin, a non-grain raw material. B. amyloliquefaciens has a highly efficient tricarboxylic acid cycle metabolic flux and glutamate synthesis ability. These features confer great potential for the synthesis of glutamate derivatives. However, it is challenging to efficiently convert high levels of glutamate to a particular glutamate derivative. Here, we conducted a systematic study on the biosynthesis of L-ornithine by B. amyloliquefaciens using inulin. First, the polyglutamate synthase gene pgsBCA of B. amyloliquefaciens NB was knocked out to hinder polyglutamate synthesis, resulting in the accumulation of intracellular glutamate and ATP. Second, a modular engineering strategy was applied to coordinate the degradation pathway, precursor competition pathway, and L-ornithine synthesis pathway to prompt high levels of intracellular precursor glutamate for l-ornithine synthesis. In addition, the high-efficiency L-ornithine transporter was further screened and overexpressed to reduce the feedback inhibition of L-ornithine on the synthesis pathway. Combining these strategies with further fermentation optimizations, we achieved a final L-ornithine titer of 31.3 g/L from inulin. Overall, these strategies hold great potential for strengthening microbial synthesis of high value-added products derived from glutamate.

Keywords: Bacillus amyloliquefaciens; L-Ornithine; L-ornithine transporter; modular metabolic engineering; non-grain raw materials.

FIGURE 1

The lack of polyglutamate synthase (pgsBCA) improves glutamate and ATP concentrations and growth. Biomass and glutamate and ATP concentrations for B. amyloliquefaciens NB, B. amyloliquefaciens (ΔpgsBCA), B. amyloliquefaciens (ΔpgsA), B. amyloliquefaciens (ΔpgsB), and B. amyloliquefaciens (ΔpgsC) in shake flasks at 24 h. All data were the average of three independent studies with standard deviations. The ** and * indicate p < 0.01 and 0.05 relative to the control strain B. amyloliquefaciens NB, respectively.

FIGURE 2

Effect of the L-ornithine synthesis module on putrescine production. (A) Schematic overview of L-ornithine catabolism pathway, precursor competition pathway, and synthesis pathways. The main pathway of l-ornithine catabolism is catalyzed by L-ornithine carbamoyltransferase and l-ornithine decarboxylase. Glutamate is a key starting material for L-ornithine synthesis. Blocking proline synthesis improves the supply of the precursor glutamate for L-ornithine synthesis. Some key genes in the L-ornithine synthesis pathway included amino-acid N-acetyltransferase (ArgA), acetylglutamate kinase (ArgB), N-acetyl-gamma-glutamyl-phosphate reductase (ArgC), acetylornithine aminotransferase (ArgD), and acetylornithine deacetylase (ArgE). G6P: Glucose-6-Phosphate; F6P: Frucose-6- Phosphate; GAP: Glyceraldedyde-3-phosphate; 1,3BPG: 1,3-Bisphospho-glyerate; 3 PG: 3-phosphoglycerate; PEP: Phosphoenolpyruvate (B) The L-ornithine titers of the recombinant strains lacking argF, argI, speF, and prob. All data were the average of three independent studies with standard deviations. The ** and * indicate p < 0.01 and 0.05 relative to the control strain B. amyloliquefaciens NB (ΔpgsBCA), respectively. (C) Overexpression of the L-ornithine synthase gene in B. amyloliquefaciens NBO6. “+” indicates that the relevant gene is overexpressed in the strain; “-” indicates that the relevant gene is not overexpressed in the strain. All data were the average of three independent studies with standard deviations. The ** and * indicate p < 0.01 and 0.05 relative to the control strain B. amyloliquefaciens NBO6, respectively. Co-expression of genes with noticeable effect to further improve the fermentation effect.

FIGURE 3

The expression of lysE increases L-ornithine synthesis and cell growth. The effect of the overexpression of four transporter genes on L-ornithine production and cell growth. Four L-ornithine transporters from different sources were screened, namely, E. coli, C. glutamicum, B. subtilis, and B. amyloliquefaciens. All data were the average of three independent studies with standard deviations. The ** and * indicate p < 0.01 and 0.05 relative to the control strain B. amyloliquefaciens NBO6, respectively.

FIGURE 4

The effect of different fermentation conditions on L-ornithine production and response surface optimization results. Three-factor and three-level response surface optimization for three major components (inulin, peptone, and MgSO₄) was done. (A) The response surface plot showed the effects of MgSO₄ and peptone on l-ornithine production. (B) The response surface plot showed the effects of inulin and peptone on L-ornithine production. (C) The response surface plot showed the effects of inulin and MgSO₄ on L-ornithine production.

FIGURE 5

The time curve of L-ornithine fermentation in a 7.5 L fermenter. (A) Time profile of single-batch L-ornithine fermentation using inulin as the carbon source in a 7.5 L fermenter. (B) Time profile of single-batch L-ornithine fermentation using inulin and sodium glutamate as the carbon source in a 7.5 L fermenter. (C) Time profile of fed-batch L-ornithine fermentation in a 7.5 L fermenter. Fermentation conditions are as follows: the working volume is 3.5-L, the stirring rate is 400 rpm, the volume air per volume is 1 vvm, and the inoculation size is 6%.