Abstract
Bio-refining lignocellulose could provide a sustainable supply of fuels and fine chemicals; however, the challenges associated with the co-utilization of xylose and glucose typically compromise the efficiency of lignocellulose conversion. Here we engineered the industrial yeast Ogataea polymorpha (Hansenula polymorpha) for lignocellulose biorefinery by facilitating the co-utilization of glucose and xylose to optimize the production of free fatty acids (FFAs) and 3-hydroxypropionic acid (3-HP) from lignocellulose. We rewired the central metabolism for the enhanced supply of acetyl-coenzyme A and nicotinamide adenine dinucleotide phosphate hydrogen, obtaining 30.0 g l−1 of FFAs from glucose, with productivity of up to 0.27 g l−1 h−1. Strengthening xylose uptake and catabolism promoted the synchronous utilization of glucose and xylose, which enabled the production of 38.2 g l−1 and 7.0 g l−1 FFAs from the glucose–xylose mixture and lignocellulosic hydrolysates, respectively. Finally, this efficient cell factory was metabolically transformed for 3-HP production with the highest titer of 79.6 g l−1 in fed-batch fermentation in mixed glucose and xylose.
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Acknowledgements
We would like to thank the Energy Biotechnology Platform of DICP for providing facility assistance. This study was financially supported by the National Natural Science Foundation of China (22161142008 and 21922812) and a DICP innovation grant (DICP I202210) from the Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS).
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J.G. and Y.J.Z. conceived the study. J.G. and W.Y. designed and performed most of the experiments. Y.L. performed fermentation experiments. M.J. prepared lignocellulosic hydrolysates. J.G., L.Y. and Y.J.Z. wrote, reviewed and revised the manuscript.
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A patent application (application no. 202310229165.5), for protecting the production of free fatty acids and 3-hydroxypropionic acid from glucose and xylose in O. polymorpha, has been filed by the Dalian Institute of Chemical Physics, Chinese Academy of Sciences, with Y.J.Z., J.G. and W.Y. named as inventors.
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Extended data
Extended Data Fig. 1 Metabolic rewiring for enhanced FFA production in O. polymorpha.
FA production requires enhanced supply of cofactor NADPH and precursor acetyl-CoA. To promote NADPH supply, pentose phosphate pathway (ZWF1) and gluconeogenesis (FBP1 and PCK1) are adopted. To promote acetyl-CoA supply, three independent strategies, including a modifiedβ-oxidation (pex10Δ), citrate lysis (MmACL, ScYHM2, AMPD), and PDH mutant (PDHm) were adopted, coupled with the overexpression of gene ACC1 to enhance malonyl-CoA supply.
Extended Data Fig. 2 Fluorescence subcellular localization of peroxisomes in control strain and pex10Δ strain.
GFP with the signal peptide SKL was targeted to peroxisomes in control strain and pex10Δ strain. Engineered strains were cultivated in minimal medium with 20 g/L glucose for 48 h, and cells were collected. GFP fluorescence in control strain was much focused, demonstrating the complete and functional peroxisomes. In contrast, GFP fluorescence in pex10Δ strain was partially dispersed into cytosol, demonstrating the destroyed peroxisome biogenesis for the enhanced supply of acetyl-CoA and acyl-CoA. The data result from at least two independent experiments with at least hree replicates in one experiment.
Extended Data Fig. 3 Cell growth and FFA production by the introduction of PDH complex and citrate lysis pathway.
a, The overexpressed PDH complex decreased FFA titers. Genes of pyruvate dehydrogenase (PDH) complex from E.coli, including a NADP+-dependent pyruvate dehydrogenase variant (EcLpdA*), dihydrolipoamide transacetylase (EcAceE), and dihydrolipoamide dehydrogenase (EcAceF), were introduced into O. polymorpha. b, Sketch map of citrate lysis pathway. Citrate was lysed into acetyl-CoA by a heterogeneous ATP citrate lyase from Mus musculus (MmACL), and a specific mitochondrial citrate transporter from S. cerevisiae (ScYHM2), and AMP deaminase (AMPD) to promote the accumulation of cytoplasmic citrate. c, Effects of overexpression of genes from citrate lysis on cell growth and FFA production. Removing the over-expressed gene AMPD in strain HpFA27L and HpFA42L had no significant effects on both FFA production (d) and cell growth (e). f, Over-expression of gene ScYHM2 partially hindered cell growth and decreased FFA production. The data are presented as the mean ± s.e.m. (n = 3 biologically independent samples).
Extended Data Fig. 4 Overexpression of MmACL and ScIDP2 in strain HpFA40 failed to promote both cell growth and FFA production.
The data are presented as the mean ± s.e.m. (n = 3 biologically independent samples).
Extended Data Fig. 5 Overexpression of gene ACC1 severely hindered cell growth.
An enhanced ACC1 expression by replacing its own promoter with PGAP in all engineered strains hindered cell growth (a), and decreased FFA titer (b). c, Overexpression of gene ACC1 by either PGAP or PTEF1 repressed cell growth, which indicated gene ACC1 was strictly regulated in O. polymorpha. The data are presented as the mean ± s.e.m. (n = 3 biologically independent samples).
Extended Data Fig. 6 Strengthened xylose assimilation for over-production of FFAs in O. polymorpha.
To strengthen xylose assimilation, xylose isomerases (XI) from both Piromyces Sp. (PspXI*) and Lachnoclostridium phytofermentans (LpXI*), coupled with native xylulokinase (XYL3), were introduced. Engineered strains were cultivated in minimal medium containing 10 g/L xylose, and cell growth (a), xylose utilization (b), and FFA production (c) were detected. d, Xylitol accumulation in control strain HpFA10 and engineered strain HpFA50 when cultivated in sole xylose within 132 h. e, Cofactor quantification in control strain and engineered strains when cultivated in sole xylose. Strains were cultivated at 37 °C, 220 rpm for 96 h, and data are presented as mean ± s.e.m. (n = 3 biologically independent samples).
Extended Data Fig. 7 Synchronous utilization of xylose and glucose for FFA production in O. polymorpha.
A hexose transporter mutant with a higher affinity to xylose (HXT1*) was introduced to strain HpFA50 under control of PGAP (HpFA53) and PPGD1 (HpFA54). On this basis, FBP1-ZWF1 were over-expressed in strain HpFA50, HpFA53, and HpFA54, generating strain HpFA55, HpFA56, and HpFA57, respectively, to enhance the supply of NADPH. All engineered strains were cultured in 10 g/L xylose (X10) to measure the cell growth (a) and xylose utilization (b). FFA titers were measured after 120 h of cultivation at 37 °C, 220 rpm (c). On the other hand, a mixture of 20 g/L glucose and 10 g/L xylose (G20X10) was adopted to conduct the batch fermentation in flasks, demonstrating cell growth (d), and the synchronous utilization of glucose (e) and xylose (f). Data are presented as mean ± s.e.m. (n = 3 biologically independent samples).
Extended Data Fig. 8 Cell growth and sugar utilization of strain HpFA58 in glucose, or/and xylose.
To promote fermentative performances, gene KU80, URA3, and LEU2 were supplemented into strain HpFA56, generating strain HpFA58. Fermentations were carried out in 20 g/L glucose (a), 10 g/L xylose (b), 20 g/L glucose+10 g/L xylose (c), to measure cell growth and xylose utilization. Strains were cultivated at 37 °C, 220 rpm for 72 h, and data are presented as mean ± s.e.m. (n = 3 biologically independent samples).
Extended Data Fig. 9 Metabolic transforming of O. polymorpha from FFA to 3-HP.
a, To recover FAA1 gene, its native terminator was replaced by TADH1 by using CRISPR-Cas9 technique. Subsequently, gene MCR under control of promoter PGAP was integrated into 4NS7 locus. PFAA1: FAA1 promoter; TFAA1: FAA1 terminator; TADH1: ADH1 terminator; FAA1dw: downstream homologous arm of FAA1 terminator; PGAP: GAP promoter; TDIT1: DIT1 terminator; 4NS7up: upstream homologous arm of 4NS7 locus; 4NS7dw: downstream homologous arm of 4NS7 locus. b, 3-HP was produced in strain XFM and GFM from 20 g/L xylose and 20 g/L glucose in minimal medium, respectively, after 72 h of cultivation at 37 °C, 220 rpm. Data are presented as mean ± stand deviation. (n = 3 biologically independent samples).
Extended Data Fig. 10 Flowchart of O. polymorpha strain construction for FFA and 3-HP production.
O. polymorpha with a disrupted gene faa1Δ was adopted for FFA accumulation, and the supply of acetyl-CoA and NADPH was enhanced for FFA overproduction. Subsequently, the xylose uptake and catabolism were enhanced for the simultaneous co-utilization of glucose and xylose. Metabolic transforming strategy was introduced to convert the FFA producing strain to 3-HP production. Gene in blue indicated the specific genetic manipulation during the metabolic engineering route (red arrow).
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Gao, J., Yu, W., Li, Y. et al. Engineering co-utilization of glucose and xylose for chemical overproduction from lignocellulose. Nat Chem Biol 19, 1524–1531 (2023). https://doi.org/10.1038/s41589-023-01402-6
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DOI: https://doi.org/10.1038/s41589-023-01402-6
