Conversion of CO2 into organic acids by engineered autotrophic yeast

Abstract

The increase of CO₂ emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO₂ as a feedstock gains an extensive importance to establish CO₂-neutral or CO₂-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO₂ as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. ¹³C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO₂ as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L^-1, while balancing the expression of key genes and modifying screening conditions led to 2 g L^-1 itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO₂ uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO₂, to support sustainability of industrial bioprocesses.

Keywords: carbon capture; metabolic engineering; organic acids; synthetic biology; yeast.

Fig. 1.

Expression of cadA and ldhL enables organic acid production in synthetic autotrophic K. phaffii. (A–D) Schematic pathways. (A) In wild-type K. phaffii methanol is oxidized to formaldehyde (black arrow) and assimilated in the XuMP cycle (orange arrows) or dissimilated to CO₂, respectively (purple arrow). (B) synthetic autotrophy in K. phaffii: the native assimilatory branch of methanol utilization was interrupted by deleting DAS1 and DAS2 (dashed gray line). AOX1 was knocked out to reduce the rate of formaldehyde formation which could be toxic to the cells. RuBisCO and PRK were integrated to complete a functional CBB cycle (green arrows). Additionally, two bacterial chaperones, groEL and groES, were overexpressed to assist the folding of RuBisCO. TDH3, PGK1, TKL1, TPI1 carrying each a peroxisomal targeting signal were overexpressed to assure the localization of the entire CBB cycle in peroxisomes. More details about the engineering strategy can be found in ref. (13). (C) Itaconic acid (red) and (D) lactic acid production (blue), (E) growth profiles, and (F) organic acid production profiles of the producing strains and the control. Time axis corresponds to the production phase under autotrophic conditions. At least three biological replicates were used in the screening to monitor the producing strains. Shades represent the SDs (±). 3PG: 3-phosphoglycerate, AcCoA: acetyl-coenzyme A, AOX1 and AOX2: alcohol oxidase 1 and 2, cadA: cis-aconitate decarboxylase, CBB cycle: Calvin-Benson-Bassham cycle, CISA_c: cytosolic cis-aconitate, CISA_m: mitochondrial cis-aconitate, DAS1 and DAS2: dihydroxyacetone synthase 1 and 2, DHA: dihydroxyacetone, FAL: formaldehyde, G3P: glyceraldehyde 3-phosphate, ITA: itaconic acid, LA: lactic acid, ldhL: L-lactate dehydrogenase, MeOH: methanol, mttA: mitochondrial tricarboxylic acid transporter, NAD⁺/NADH: nicotinamide adenine dinucleotide, PRK: phosphoribulokinase, PYR: pyruvate, RuBP: ribulose 1,5-bisphosphate, RuBisCO: ribulose 1,5-bisphosphate carboxylase/oxygenase, Xu5P: xylulose 5-phosphate, XuMP cycle: xylulose monophosphate cycle.

Fig. 2.

Fine-tuning the balance of expression of cadA and mttA increases itaconic acid production. (A) Growth profiles, (B) itaconic acid production profiles of the itaconic acid producing strain and the control. Time axis corresponds to the production phase under autotrophic conditions. At least three biological replicates were used in the screening to monitor the producing strains. Shades represent the SDs (±).

Fig. 3.

Deletion of CYB2 prevents lactic acid consumption. (A) Growth profiles, (B) lactic acid consumption profiles of the lactic acid producing strain, the lactic acid producing cyb2Δ strain, and the cyb2Δ strain. Time axis corresponds to the cultivation time on media containing lactic acid under nonautotrophic conditions. At least three biological replicates were used in the screening to monitor the producing strains. Shades represent the SDs (±).

Fig. 4.

Deletion of CYB2 improves lactic acid production in synthetic autotrophic K. phaffii. (A) Growth profiles, (B) lactic acid production profiles, (C) glycolic acid production profiles of the lactic acid producing strain and the control. Time axis corresponds to the production phase under autotrophic conditions. At least three biological replicates were used in the screening to monitor the producing strains. Shades represent the SDs (±).

Fig. 5.

Reverse labeling confirms that carbon in lactic and itaconic acid is incorporated from the captured CO₂. (A) Experimental design of the reverse labeling approach. After labeling the cells on ¹³C glycerol, we inoculated them into YNB and supplemented them with ^natC CO₂ (CO₂ with natural isotope distribution) and ¹³C MeOH. Samples were taken at different time points (day 3, day 5 and day 8) for the analysis with GC-TOFMS to monitor the change in the¹³C labeling pattern of the produced organic acids. (B) ¹³C labeling degree at different timepoints of the produced itaconic acid using the production strain on the left and the nongrower control. (C) ¹³C labeling degree at different timepoints of the produced lactic acid using the production strain on the left and the nongrower control. Error bars indicate the SD of 3 biological replicates. Detailed isotopologue distribution of the carbon atoms can be found in SI Appendix, Fig. S5.

Fig. 6.

Modification of process conditions shows that each product has its own requirements for an optimum bioprocess. (A, B) Itaconic acid production profiles and (C, D) lactic acid production profiles at 5% CO₂ and 10% CO₂ supply, respectively, of producing clones and the control. Time axis corresponds to the production phase under autotrophic conditions. At least two biological replicates were used in the screening to monitor the producing strains. Shades represent the SDs (±).

Fig. 7.

Bioreactor cultivations show each process should be designed based on the specific requirements of the target product. (A) Growth and (B) itaconic acid production profile at constant DO concentrations 8% and 20%. (C) Growth, (D) lactic, and (E) glycolic acid production profile at 21% and 5% inlet oxygen concentrations. Time axis corresponds to the production phase under autotrophic conditions. Bioreactor cultivations for itaconic acid production were done in duplicates and shades represent the SD (±). For lactic acid results of each fermentation run are shown separately.