Production of Biopolyamide Precursors 5-Amino Valeric Acid and Putrescine From Rice Straw Hydrolysate by Engineered Corynebacterium glutamicum

Abstract

The non-proteinogenic amino acid 5-amino valeric acid (5-AVA) and the diamine putrescine are potential building blocks in the bio-polyamide industry. The production of 5-AVA and putrescine using engineered Corynebacterium glutamicum by the co-consumption of biomass-derived sugars is an attractive strategy and an alternative to their petrochemical synthesis. In our previous work, 5-AVA production from pure xylose by C. glutamicum was shown by heterologously expressing xylA from Xanthomonas campestris and xylB from C. glutamicum. Apart from this AVA Xyl culture, the heterologous expression of xylA _Xc and xylB _Cg was also carried out in a putrescine producing C. glutamicum to engineer a PUT Xyl strain. Even though, the pure glucose (40 g L^-1) gave the maximum product yield by both the strains, the utilization of varying combinations of pure xylose and glucose by AVA Xyl and PUT Xyl in CGXII synthetic medium was initially validated. A blend of 25 g L^-1 of glucose and 15 g L^-1 of xylose in CGXII medium yielded 109 ± 2 mg L^-1 putrescine and 874 ± 1 mg L^-1 5-AVA after 72 h of fermentation. Subsequently, to demonstrate the utilization of biomass-derived sugars, the alkali (NaOH) pretreated-enzyme hydrolyzed rice straw containing a mixture of glucose (23.7 g L^-1) and xylose (13.6 g L^-1) was fermented by PUT Xyl and AVA Xyl to yield 91 ± 3 mg L^-1 putrescine and 260 ± 2 mg L^-1 5-AVA, respectively, after 72 h of fermentation. To the best of our knowledge, this is the first proof of concept report on the production of 5-AVA and putrescine using rice straw hydrolysate (RSH) as the raw material.

Keywords: 5-amino valeric acid; Corynebacterium glutamicum; polyamides; putrescine; rice straw hydrolysate.

FIGURE 1

Schematic representation of the putrescine production route in Corynebacterium glutamicum PUT Xyl (A) and the 5-aminovalerate production route in C. glutamicum AVA Xyl (B). Gene names of key enzymes are given next to the corresponding reaction (pointed arrows). Black-boxed gene names indicate plasmid-borne expression, unboxed gene names indicate genomic expression. Reaction routes involving more than one gene are depicted by dashed arrows. Repression is represented by blunt arrows. Upregulated reactions are shown in green, downregulated reactions are shown in blue, and deleted reactions are shown in red. The green-boxed arginine pathway in (A) indicate upregulation by deletion of the repressor of the arginine biosynthesis ArgR. argB^A49V/M54V, feedback-resistant N-acetyl glutamate kinase; argF, ornithine transcarbamoylase; argF₂₁, ornithine transcarbamoylase with reduced activity; argR, repressor of the arginine biosynthesis; asd (2 copies), aspartate-semialdehyde dehydrogenase; cgmA, cyclic beta-1,2-glucan modification protein; dapA (2 copies), 4-hydroxy-tetrahydrodipicolinate synthase; dapB (2 copies), 4-hydroxy-tetrahydrodipicolinate reductase; ddh (2 copies), meso-diaminopimelate D-dehydrogenase; gabD, succinate-semialdehyde dehydrogenase; gabT, 4-aminobutyrate aminotransferase; gapA, Glyceraldehyde-3-phosphate dehydrogenase A; hom^V59A, reduced activity homoserine dehydrogenase; ldcC, constitutive lysine decarboxylase; ldhA, D-lactate dehydrogenase; lysA (2 copies), diaminopimelate decarboxylase; lysC^T311I (2 copies), high activity aspartokinase; lysE (2 copies), lysine exporter; odhA^TTG, reduced activity 2-oxoglutarate dehydrogenase; odhI^T15A, high activity oxoglutarate dehydrogenase inhibitor; patA, putrescine aminotransferase; patD, gamma-aminobutyraldehyde dehydrogenase; pck, phosphoenolpyruvate carboxykinase; P_tac, IPTG-inducible tac promoter; pyc, pyruvate carboxylase; pyc^P458S, high activity pyruvate carboxylase; snaA, N-acetyltransferase; speC, ornithine decarboxylase; sugR, central transcriptional regulator of the carbon metabolism; xylA_Xc, xylose isomerase from Xanthomonas campestris; xylB_Cg, xylulose kinase from C. glutamicum.

FIGURE 2

Putrescine production of C. glutamicum PUT Xyl. Growth curves of C. glutamicum PUT Xyl cultivated in shake flasks of 20 mL CGXII minimal medium supplemented with different compositions of glucose (Gluc) and xylose (Xyl) (A), and putrescine concentrations measured after 0, 6, 24, and 72 h of cultivation (B). Measurements are given as means from triplicates of independent cultivations with standard deviation.

FIGURE 3

Production of putrescine in CGXII synthetic medium with different blends of glucose and xylose. The percentage utilization of glucose and xylose by PUT Xyl is shown in (A). The glucose and xylose consumption rate of PUT Xyl is shown in (B). The time profile of the putrescine production is shown in (C). Measurements are given as means from triplicates of independent cultivations with standard deviation.

FIGURE 4

Production of 5-AVA in CGXII medium with different blends of glucose and xylose. The utilization of glucose and xylose by PUT Xyl is shown in (A). The glucose and xylose consumption rate of AVA Xyl is shown in (B). The time profile of the 5-AVA production is shown in (C). Measurements are given as means from triplicates of independent cultivations with standard deviation.

FIGURE 5

Concentration of glucose and xylose in RSH (A). Production of putrescine (B) and 5 AVA (C) from RSH. Hydrolysate contained 2.37 g L^–1 glucose + 12.6 g L^–1 xylose. The downward diagonal column represents the production and the line graph with marker shows the absorbance at OD 600 nm. Measurements are given as means from triplicates of independent cultivations with standard deviation.