Metabolic engineering to improve production of 3-hydroxypropionic acid from corn-stover hydrolysate in Aspergillus species

Abstract

Background Fuels and chemicals derived from non-fossil sources are needed to lessen human impacts on the environment while providing a healthy and growing economy. 3-hydroxypropionic acid (3-HP) is an important chemical building block that can be used for many products. Biosynthesis of 3-HP is possible; however, low production is typically observed in those natural systems. Biosynthetic pathways have been designed to produce 3-HP from a variety of feedstocks in different microorganisms. Results In this study, the 3-HP β-alanine pathway consisting of aspartate decarboxylase, β-alanine-pyruvate aminotransferase, and 3-hydroxypropionate dehydrogenase from selected microorganisms were codon optimized for Aspergillus species and placed under the control of constitutive promoters. The pathway was introduced into Aspergillus pseudoterreus and subsequently into Aspergillus niger, and 3-HP production was assessed in both hosts. A. niger produced higher initial 3-HP yields and fewer co-product contaminants and was selected as a suitable host for further engineering. Proteomic and metabolomic analysis of both Aspergillus species during 3-HP production identified genetic targets for improvement of flux toward 3-HP including pyruvate carboxylase, aspartate aminotransferase, malonate semialdehyde dehydrogenase, succinate semialdehyde dehydrogenase, oxaloacetate hydrolase, and a 3-HP transporter. Overexpression of pyruvate carboxylase improved yield in shake-flasks from 0.09 to 0.12 C-mol 3-HP C-mol−1 glucose in the base strain expressing 12 copies of the β-alanine pathway. Deletion or overexpression of individual target genes in the pyruvate carboxylase overexpression strain improved yield to 0.22 C-mol 3-HP C-mol−1 glucose after deletion of the major malonate semialdehyde dehydrogenase. Further incorporation of additional β-alanine pathway genes and optimization of culture conditions (sugars, temperature, nitrogen, phosphate, trace elements) for 3-HP production from deacetylated and mechanically refined corn stover hydrolysate improved yield to 0.48 C-mol 3-HP C-mol−1 sugars and resulted in a final titer of 36.0 g/L 3-HP. Conclusions The results of this study establish A. niger as a host for 3-HP production from a lignocellulosic feedstock in acidic conditions and demonstrates that 3-HP titer and yield can be improved by a broad metabolic engineering strategy involving identification and modification of genes participated in the synthesis of 3-HP and its precursors, degradation of intermediates, and transport of 3-HP across the plasma membrane.

Metabolic engineering in lignocellulose biorefining for high-value chemicals: recent advances, challenges, and outlook for enabling a bioeconomy

“…However, there are limited reports on the use of LCB as a feedstock for 3-HP production (Table 2). In a study conducted by Dai et al (2023) the production of 3-HP from corn stover biomass was investigated. They constructed a recombinant Aspergillus niger by introducing 12 copies of the b−alanine pathway (constructed by including Tribolium castaneum panD, B. cereus bapat, and E. coli hpdh under the control of constitutive gpdA promoter).…”

“…The final engineered strain could produce 29.4 g/L of 3-HP with a yield of 0.35 mole/mole sugars from corn stover hydrolysate. When an additional 15 copies of the b−alanine pathway were introduced under the promoter of ubi4 and mbfA along with optimization of the fermentation condition, the 3-HP production improved to 36 g/ L with a yield of 0.48 mole/mole sugars (Dai et al, 2023). In another study, Liu et al (2023) focused on the utilization of the malonyl-CoA pathway for the production of 3-HP from corn stover hydrolysate.…”

Evaluation of OxiOrganosolv pretreated hardwood and softwood lignocelluloses as substrates for the chemoenzymatic production of 5-hydroxymethylfurfural (HMF)

“…Among popular fields are the synthesis of chemicals, food additives, pharmaceuticals, and polymeric materials [2,3]. In particular, many biobased-polymers from lignocellulose have been developed, including nanocellulose, polylactic acid (PLA), and polyhydroxyalkanoates (PHAs), such as poly(3-hydroxypropionic acid) and polyhydroxybutyrate, by combining microbial fermentation and enzyme-mediated processes with chemical catalysis [4][5][6][7][8]. In this way, lignocellulosic biomass can serve as a substrate for the synthesis of green plastics, thus lowering the dependence of using petrochemicals and fossil fuels.…”

Novel genetic tools improve Penicillium expansum patulin synthase production in Aspergillus niger

“…The effectiveness of the production is governed by many endogenous and exogenous factors, such as various regulatory proteins or metabolites inside the producing cells. To achieve optimal production, multiomics data would need to be generated from diverse culture conditions and genotypes [13]. Such omics data would form the foundation to understanding, via machine learning, how the endogenous factors exert their effects on the production of a specific protein or chemical.…”