Methanol biotransformation for the production of biodegradable plastic monomer L-lactate in yeast

Domain: physics
Published: 2025-11-28
Editorial outcome: major_revision

Abstract
Methanol is an ideal feedstock for biomanufacturing, and production of the biodegradable plastic monomer lactate from methanol is a promising approach for mitigating white pollution. However, it is challenging to engineer microbes for lactate production from methanol because of strong competition between product synthesis and cell growth. Here, Ogataea polymorpha is rewired to synthesize L-lactate from methanol, where the cofactor ratio of NADPH/NADP+ is higher than that of NADH/NAD+. By engineering gene expression, enhancing cell viability, modifying cofactor homeostasis, and performing mitochondrial compartmentalization, 2.5 g/L L-lactate is produced in a shake flask. Fed-batch fermentation in a bioreactor results in the highest titer of 25.0 g/L L-lactate from methanol, which is chemically synthesized from CO2. A techno-economic analysis and life cycle assessment are performed to evaluate the commercial potential, environmental impacts, and greenhouse gas mitigation performance of CO2-derived L-lactate. This study could lay the foundation for the carbon-neutral production of biodegradable plastic poly-lactic acid from CO2.

Introduction
A world without plastics seems unimaginable, as plastics are widely used in food packaging, agriculture, and transportation, among other industries1. However, fossil fuel-derived commodity plastics, such as polyethylene (PE), polypropylene (PP), and polyvinyl chloride (PVC), are undegradable, which causes severe white pollution. When these plastics are fragmented into small pieces, known as microplastics, the risks to human health increase2. To address the problems of undegradable plastics and the heavy reliance on fossil fuels, biobased degradable plastics, among which polylactic acid (PLA) is the most widely used, have attracted considerable attention. PLA is industrially synthesized through ring-opening polymerization of lactide, which is obtained from the monomer lactate. Chemical synthesis of lactate uses petrochemical feedstocks and results in racemic mixtures of D/L-lactate3. In contrast, bioproduction generates enantiomerically pure lactic acid (D- or L-form), which facilitates the synthesis of poly(D-lactic acid), poly(L-lactic acid), and poly(DL-lactic acid), which have different properties4.

Biorefineries can be divided into three generations: the 1st generation (1G) uses glucose as feedstocks; the 2nd generation (2G) uses nonfood lignocellulosic biomass as feedstocks; and the 3rd generation (3G) uses one carbon (C1) source, such as CO2, methane, or methanol, as feedstocks5. Although major companies, including NatureWorks and BASF, have employed 1G feedstocks for lactate production with high yields (0.9 g/g)6, the large demand for PLA may suffer from competition with the food supply. 2G biorefinery has helped promote progress in lactate production from non-edible biomass feedstocks, but complex pretreatment and enzymatic saccharification still hinder profitability7. In the recent decade, we have witnessed a growing emphasis on 3G biorefineries, including starch synthesis from CO2 8; the production of single-cell proteins from methane9 or methanol10; and the production of chemicals, such as fatty acids, fatty alcohols, and 3-hydroxypropionate, from methanol11–15. C1-fueled bioprocesses are independent of arable land and can mitigate greenhouse effects; thus, C1-fueled bioprocesses are promising bioprocesses for lactate production.

Among C1 feedstocks, methanol has many advantages, including convenient storage and transportation, efficient liquid-to-liquid mass transfer, and high reducing power16. In particular, methanol can be massively produced from CO2 via liquid sunshine processes (CO2 hydrogenation to methanol)17. Therefore, coupling liquid sunshine and methanol bioconversion enables the carbon-negative production of chemicals from CO2. However, the toxicity of methanol and its rigid metabolism limit the efficiency of chemical production12,18,19. Although many endeavors have been made to either engineer native methylotrophs such as Pichia pastoris and Ogataea polymorpha or construct synthetic methylotrophs such as Escherichia coli, the titers of lactate from methanol are far from adequate for commercialization. The main reason may be the trade-off between methanol metabolism and lactate synthesis, which compete not only for carbon flux but also for the energy supply, especially the cofactor NADH.

Here, we observe a pattern of cofactor distribution during methanol metabolism in the methylotrophic yeast O. polymorpha, which is an industrial strain with strong osmotic and thermal tolerance (reaching 50 °C)20,21. Although methanol metabolism is relatively rigid, the global metabolic redesign of O. polymorpha, which requires optimizing the gene expression of lactate dehydrogenase (Ldh), enhancing cell viability, engineering cofactor homeostasis, and performing mitochondrial compartmentalization, greatly increases L-lactate synthesis from sole methanol. Coupling liquid sunshine and methanol biotransformation achieved economic viability and enabled the carbon-negative production of L-lactate from CO2, which was evaluated via techno-economic analysis (TEA) and life cycle assessment (LCA). This study enables the production of PLA from CO2, a recycling economy that connects biodegradable plastics and carbon neutrality (Fig. 1).

Results
Constructing an L-lactate biosynthetic pathway from methanol alone
Lactate can be produced from an intermediate pyruvate in a central metabolism. This material is catalyzed by lactate dehydrogenase (Ldh) with NADH as a cofactor. In nature, lactic acid bacteria (LAB) can synthesize lactate from glucose with a high yield of 0.9 g/g due to the high efficiency of Ldh6. Here, the methylotrophic yeast O. polymorpha, which cannot produce native lactate, was adopted to produce L-lactate from methanol (Fig. 2a). Five different L-LDH genes from LAB (Supplementary Table 1) were evaluated for L-lactate production by using the methanol-inducible promoter PAOX1 in an episomal plasmid. PaLdh from Pediococcus acidilactici behaved the best among the tested specimens in O. polymorpha (strain HpPa), with 921 mg/L L-lactate being produced from 10 g/L methanol (Fig. 2b; LC‒MS determination in Supplementary Fig. 1). Optical isomer detection by HPLC revealed that the optical purity of L-lactate was >99% (Supplementary Fig. 2). Lactobacillus delbrueckii LdLdh and Lactobacillus helveticus LhLdh exhibited no L-lactate production (Fig. 2b), which, however, showed functional expression at the transcriptional level (Supplementary Fig. 3a). To ensure a stable and optimal expression level, PaLDH was integrated into the genome at different neutral sites with different promoters22,23. Five promoters were characterized by using enhanced green fluorescent protein (eGFP), which revealed that PAOX1 and PGAP were the strongest -inducible and constitutive promoters in methanol, respectively (Supplementary Fig. 3b). Despite variable expression levels of different neutral sites, as previously described22, the production of L-lactate from methanol was affected mainly by promoters rather than neutral sites (Fig. 2c). Strain 3A, which expressed PaLDH under the control of the PAOX1 promoter at the NS3 site, produced 655 mg/L L-lactate. To further promote cell fitness22,24, the LEU2 gene was recovered in situ in situ (Supplementary Fig. 4) from the strains HpPa and 3A, generating strains HPLA05 and HPLA06, respectively. Interestingly, strain HPLA06 produced 1.2 g/L L-lactate, with levels 25% greater than those of HPLA05 (Fig. 2d), demonstrating that one copy of PaLDH in the genome was sufficient to outperform multiple copies in the plasmid. Therefore, the engineered strain HPLA06 was used as the parent strain for further investigation.

Enhancing cell viability in methanol
L-lactate synthesis strongly competes with the tri-carboxylic acid (TCA) cycle for pyruvate25, resulting in a growth delay in methanol. We observed that the biomass of HPLA06 with 1.2 g/L L-lactate dramatically decreased relative to that of the wild-type strain (Supplementary Fig. 5). We evaluated the L-lactate tolerance of O. polymorpha, which showed that 5 g/L L-lactate exhibited no growth inhibition to O. polymorpha (Supplementary Fig. 6). This phenomenon prompted us to explore other strategies to promote cell robustness to increase L-lactate production.

Our previous study demonstrated that phospholipid homeostasis was essential for cell survival in methanol through the inactivation of LPL1 (encoding a putative lipase) and IZH3 (encoding a membrane protein)12. Therefore, single or double deletions of LPL1 and IZH3 were performed in HPLA06, and the results revealed that IZH3 deletion alone improved the L-lactate titer by 23%, with a similar growth profile, whereas LPL1 deletion negatively impacted L-lactate production (Fig. 3a). To determine the mechanism of improved L-lactate synthesis in the IZH3 deletion strain, propidium iodide (PI) staining26,27 was used to determine the cell death rate (PI-positive). As expected, L-lactate production caused more than 10% of the cells to die within 24 h, whereas IZH3 deletion marginally decreased the cell death rate to approximately 5% (Fig. 3b and Supplementary Fig. 7), thus increasing cell viability to promote L-lactate synthesis. In this case, we attempted to employ adaptive laboratory evolution (ALE) to further improve the cell growth of the engineered strain HPLA06 in methanol. Unfortunately, the L-lactate titer decreased by 62% after 8 rounds of transfer, despite the 128% increase in biomass (Supplementary Fig. 8). Therefore, ALE decreased L-lactate production from methanol, and the mechanism remains to be explored in the future.

Engineering cofactor homeostasis to improve L-lactate synthesis
To promote the growth of L-lactate-producing cells, we believed that cofactor imbalance could be a limiting factor since L-lactate synthesis requires equal amounts of precursor pyruvate and the cofactor NADH (Fig. 4a). Glucose metabolism is conducted mainly through glycolysis, which generates NADH, whereas methanol assimilation is conducted via the pentose phosphate pathway (PPP) and xylulose-5-phosphate (Xu5P) cycle, which generate NADPH (Fig. 4a). We found that the cofactor NADPH was more abundant in methanol than in glucose (Fig. 4b). Hence, the cofactor preference of PaLdh was engineered from NADH to NADPH by a single-point mutation at the cofactor binding site (V40R)28,29 (Supplementary Fig. 9). However, one copy integration of PaLDHV40R dramatically decreased L-lactate production (Fig. 4c), probably owing to its relatively low activity. Another copy of PaLDHV40R (strain HPLA13) resulted in 31% higher L-lactate production than did the control strain (strain HPLA07) with native PaLDH (Fig. 4c). However, the third PaLDHV40R copy (strain HPLA17) decreased both cell growth and L-lactate production (Supplementary Fig. 10). In addition, cofactor determination revealed that strain HPLA13, with two copies of NADPH-dependent PaLDHV40R, could achieve cofactor balance, differing from observation in the L-lactate-producing strain with NADH-dependent PaLDH (Fig. 4d). In particular, NADPH-dependent PaLdh did not promote L-lactate synthesis during glucose cultivation (Fig. 4e), considering the similar ratios of NADPH/NADP+ and NADH/NAD+ (Fig. 4b), which demonstrated the trait of a relatively high NADPH/NADP+ ratio during methanol metabolism for product synthesis. It is worth mentioning that PGAP-driven LDH constructs were used for the engineered strains to strengthen LDH gene expression in glucose cultivation. Finally, the strategy of promoting cell viability by deleting the IZH3 gene was used to obtain strain HPLA14, which produced 2.1 g/L L-lactate from 10 g/L methanol, a level 57% greater than that of strain HPLA13 (Fig. 4f). Similarly, adding a third copy of LDHV40R in HPLA14 did not further improve L-lactate production (Supplementary Fig. 11).

Mitochondrial compartmentalization of Ldh
In yeast, pyruvate can enter mitochondria to produce acetyl-CoA, which is further catabolized to yield NADH. In this case, mitochondria are likely an ideal compartment for L-lactate synthesis because of the abundant supply of pyruvate and NADH. Here, the dual-regulation of cytoplasmic and mitochondrial engineering for L-lactate production was carried out by simultaneously expressing NADPH-dependent Ldh in the cytoplasm and NADH-dependent Ldh in mitochondria (Fig. 5a). For mitochondrial localization, an N-terminal mitochondrial targeting signal (MTS) from subunit IV of yeast cytochrome oxidase (CoxIV)30 was selected for fusion with eGFP. Fluorescence microscopy revealed the correct mitochondrial localization of the MTS-fused eGFP by using the mitochondrial marker Mito-tracker Red as a reference (Fig. 5b). MTS was subsequently used to target NADH-dependent PaLdh into mitochondria under the control of the growth phase-dependent promoter Pro-Y1331. Integration of the PaLDH gene into the NS5 and NS6 loci resulted in 7% and 22% increases in L-lactate production, respectively (Fig. 5c). The engineered strain HPLA22 produced 2.5 g/L L-lactate, with a yield of 0.25 g/g methanol. Further increasing the copy number of PaLDH did not promote product synthesis (Supplementary Fig. 12).

Fed-batch fermentation in bioreactors for L-lactate production
To enable stable L-lactate production, the URA3 gene was recovered in situ from strain HPLA22, which marginally increased the L-lactate titer (Supplementary Fig. 4 and Supplementary Fig. 12). To evaluate the potential of carbon-negative L-lactate production, liquid sunshine technology (chemical catalysis of CO2 to methanol)32 and methanol biotransformation (bioconversion of methanol to L-lactate) were combined. The CO2-derived methanol was a solution of methanol and water with a ratio of 2.25:1 (v/v). The high methanol concentration of 593 g/L without impurities was suitable for methanol feeding. The prototrophic strain HPLA22u was subsequently used for fed-batch fermentation with CO2-derived methanol as the sole carbon and energy source. After 252 h of fermentation, 114 g/L methanol was consumed, resulting in the production of 25.0 g/L L-lactate, with a yield of 0.22 g/g (Fig. 5d).

TEA and LCA of L-lactate biomanufacturing derived from CO2
We developed a process employing CO2-derived methanol for the manufacture of L-lactate to conduct TEA and LCA for biorefinery utilizing recombinant O. polymorpha HPLA22u by Aspen plus v14 (Fig. 6a). On the basis of the production metrics and assumed parameters obtained from experimental results or the literature33–35 (Supplementary Tables 2-3), the proposed production scenario was indicative of the capacity of a plant to produce 18,500 tons of L-lactate annually through three major conversion steps, as illustrated in Supplementary Figs. 13-14, which depicts the primary carbon flow within the various subsystems and the detailed process design in Aspen plus, respectively. It is worth noting that projected production capacity corresponds with standard industrial lactic acid plant scales (15,000-30,000 tons per year)36 and is in accordance with capacities frequently utilized in published TEA studies on third-generation biomanufacturing37,38. The capital expenses (CAPEX) for this plant are described in Fig. 6b and Supplementary Table 4, including fixed capital investment (FCI), total indirect costs (TICs), and total direct costs (TDCs). Among the five modules of the process (methanol production, L-lactate production, L-lactate purification, wastewater treatment, and utilities), the L-lactate production subsystem was determined to necessitate the greatest installation cost for equipment. The CAPEX for equipment and installation of five modules was 2.3%, 59.5%, 12.3%, 7.3%, and 18.6%, respectively (Supplementary Table 4). Comprehensive investment details for equipment and installation corresponding to each individual component are available in Supplementary Fig. 15. Notably, the bioreactors accounted for the predominant share of expenditures associated with equipment and installation in CAPEX. The investment in the purification unit is less extensive compared to the traditional separation process, the membrane separation approach operates under milder conditions and involves fewer steps, hence decreasing both energy requirements and complexity39,40. In terms of operational expense (OPEX), raw materials constituted the primary factor in manufacturing costs (Fig. 6c and Supplementary Table 5). Notably, this analysis considered carbon trading as a potential credit for the entire process. The ongoing implementation of carbon tax policies and the rise in carbon trading prices markedly enhanced economic efficiency41. Furthermore, the residual biomass can be marketed as nitrogen fertilizer through anaerobic digestion. When we assumed operation of the L-lactate production process for 30 years with an internal rate of return (IRR) of 10%, the net present value (NPV) became zero at an L-lactate minimum selling price (MSP) of $2.29 kg-1, which was comparable to the prevailing market price of $1.85–3.15 kg-1 34,42. The cost distribution for the MSP of five modules was 31.2%, 43.9%, 9.5%, 3.0%, and 12.4%, respectively (Supplementary Table 6). The MSP of the baseline scenario indicated the economic viability of CO2-derived L-lactate production and its prospective ability to achieve economic competitiveness.

On the basis of the findings of CAPEX and OPEX, the costs associated with equipment, installation, feedstock, and carbon tax revenue exhibited the highest sensitivity to MSP. Consequently, we adjusted the values of eight pertinent parameters to generate the corresponding variation range for a single-point sensitivity analysis. We intended to ascertain the impacts of different parameters on the scale-up process of L-lactate derived from CO2 and to find prospective opportunities for enhanced commercial viability (Fig. 6d). Sensitivity analysis indicated that the yield of L-lactate from CO2-derived methanol at the break-even point was highly sensitive to overall costs; an increase in L-lactate yield from 0.22 to 0.26 g/g led to a 15.4% reduction in the MSP, which was chiefly attributed to the strong correlation between the increase in annual plant capacity and the MSP. Moreover, lower productivity and titer could lead to a 11.2–12.0% increase in MSP, as CAPEX significantly correlated with this parameter of annual production. To sustain the annual 18,500-ton capacity with lower productivity, increased equipment investment was needed for the acquisition of additional bioreactors. Since yield, productivity, and titer were closely linked to the carbon flux directed toward L-lactate, which impacted the overall capacity of the plant, these parameters were notable as major cost drivers. In addition, the fluctuations in the carbon source price and power costs exhibited significant effects. When the solar power price ranges from 0.02–0.04 $/kWh, the MSP varies by ±(5.0–5.9)% ($2.15–2.40 kg−1). Considering the regional variation in solar power costs, the MSP fluctuates significantly with the geographical location of the plant43 (Supplementary Fig. 16). For example, countries with abundant solar resources such as Pakistan show solar power prices close to the global minimum (<0.025 $/kWh), resulting in comparatively lower MSP, whereas regions with higher solar power prices such as Canada (>0.10 $/kWh) may increase the MSP by over 13%. These results emphasize the critical role of site selection in improving the economic competitiveness of biomanufacturing L-lactate from CO2. The findings indicated that the parameters related to L-lactate production in this study substantially influenced the economic viability of process commercialization and underscored the opportunity for cost reduction through improved carbon source conversion efficiency and reduced atmospheric CO2 capture cost. Although the MSP of the base scenario demonstrated remarkable economic performance, enhancements in fermentation-related parameters could further augment its economic feasibility.

Environmental impact assessment is a vital element influencing the viability of sustainably scaling up and commercializing the L-lactate production process. A preliminary LCA was performed to illustrate the global warming potential (GWP) linked to the production of 1 kg of L-lactate, with the lifecycle inventory (LCI) derived from the Aspen Plus simulation (Supplementary Table 7). As shown in Fig. 6e, the cradle-to-gate lifecycle GWP of 1 kg of L-lactate produced from methanol was 1.74 kg CO2-eq. The contribution analysis of GWP hotspots revealed that power consumption (52.19%), CO2 emission from combined heat and power (32.62%), and the nitrogen source for cell growth (14.62%) were the primary contributors across all impact categories (Supplementary Table 8). It is worth mentioning that from the standpoint of economic value allocation, the by-product of fertilizer could result in a GWP decrease of 0.277 kg CO2-eq, so further diminishing the carbon footprint. The results indicated that the suggested approach for L-lactate production could significantly decrease greenhouse gas (GHG) emissions through the incorporation of alternative renewable energy sources. Notably, the ammonia production process, known as Haber–Bosch, could be characterized by high energy consumption and greenhouse gas emissions due to fossil fuel combustion, contributing to the global warming potential. Nonetheless, to guarantee the appropriate development of microorganisms, this environmental influence is necessary. As a reference, the environmental impacts of the current L-lactate production method were compared with those of fossil-based refineries (produced via acetaldehyde44), and lignocellulose-based biorefineries (including lignocellulosic biomass45, biomass saccharides44, and vine shoots46) demonstrated a potential reduction in GWP reaching 2.56 tons of GHG emissions per ton of L-lactate produced (Fig. 6f). The competitive advantage of GWP principally stemmed from CO2 serving as the carbon source for process design, which is often considered a principal GHG in the atmosphere, with the synthesis of 1 ton of L-lactate resulting in a carbon deposition of 7.29 tons (Supplementary Table 8). Consequently, the biomanufacturing of L-lactate from CO2-derived methanol using O. polymorpha provides a viable approach for harnessing atmospheric GHGs to achieve low-carbon production while possessing the potential for commercial competitive business activities.

Methods
Plasmids, strains, and cultivation conditions
All codon-optimized genes and the primers used in this study are listed in Supplemental Data 1 and 2 in Source Data, respectively. All plasmids and strains used in this study are listed in Supplementary Table 10 and Supplemental Data 3 in Source Data, respectively, and the strain construction process is described in Supplementary Fig. 17. The seed culture of the yeast strain was cultivated in YPD medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L D-glucose) at 37 °C. After transformation, the recombinant strains were placed in synthetic droplet medium (6.7 g/L yeast nitrogen base and 20 g/L glucose). The gRNA plasmid containing the URA3 selection marker was removed after two rounds of transfer to YPD medium. All the solid media were supplemented with 20 g/L agar. Escherichia coli DH5α was used for plasmid transformation and cultured in Luria–Bertani medium (10 g/L peptone, 5 g/L yeast extract, and 10 g/L NaCl) supplemented with 100 mg/L ampicillin at 37 °C.

Genetic manipulation
All engineered O. polymorpha strains were constructed via the CRISPR‒Cas9 system49. Gene deletion and integration were performed according to a standard procedure: gRNA design, plasmid and donor DNA construction, transformation, colony verification, and plasmid removal13,49. Gene integration was conducted at neutral sites in the genome, as previously described22. Donor DNA, consisting of upstream homologous arm (HA), promoter, target gene, terminator, and downstream HA, was generated by using overlap extension PCR (OE-PCR). Taking NS3-PAOX1-LDH as an example, 1000 bp NS3 upstream HA, 1000 bp AOX promoter, 341 bp AMO terminator, 1000 bp NS3 downstream HA, and 972 bp PaLDH gene were PCR amplified from O. polymorpha genomic DNA and PaLDH synthetic plasmid, respectively. The donor DNA generated by OE-PCR was gel-purified and transformed with the NS3 gRNA plasmid into O. polymorpha competent cells and selected on Synthetic Droplet (SD) plates. 10 clones were selected and verified by colony PCR. After PCR verification, the correct clones were cultured overnight in YPD medium and stored at −80 °C in 20% (v/v) glycerol, followed by the next round of genetic engineering.

Batch fermentation and fed-batch fermentation
Shake flask fermentation was performed at 37 °C and 220 r/min for 96 h in Delft medium (2.5 g/L (NH4)2SO4, 14.4 g/L KH2PO4, 0.5 g/L MgSO4·7H2O, 2 mL/L trace metal, and 1 mL/L vitamin solution69) containing 10 g/L methanol. The pH was adjusted to 5.6 by using 4 mol/L KOH, and 60 mg/L uracil was added to the medium if needed. But the pH was not further adjusted during cultivation in shake flasks. After fermentation, the broth was sampled to determine the OD600 and L-lactate titer by using spectrometry and high-performance liquid chromatography (HPLC), respectively.

For fed-batch fermentation with methanol as the sole carbon source, single colonies of the engineered strain were cultivated in YPD medium for approximately 24 h and then transferred into minimal medium containing 20 g/L glucose with an initial OD600 of 0.5 for approximately 10 h. The seed cultures were inoculated into 1 L bioreactors (DasGip Parallel Bioreactor System, Eppendorf, Germany) with a working volume of 0.4 L. The pH, temperature, and dissolved oxygen (DO) were set as 5.6, 37 °C, and 30%, respectively. The pH was controlled at a constant 5.6 with the feeding 4 M KOH to neutralize the produced L-lactate. Initial agitation was set to 400 rpm, and increased to a maximum 800 rpm, depending on the DO level. Aeration was initially provided at 18 sL/h and increased to maximally 48 sL/h depending on the DO level. The initial batch fermentation was carried out in Delft medium containing 10 g/L methanol (Delft-M10). The control system was the same as previously described12, such as every 12 h, samples were collected to measure OD600, methanol, and L-lactate. Three biologically independent parallel experiments were conducted during the fermentation. The 10× Delft medium and CO2-derived methanol (593 g/L) were pumped into the vessels. The feeding rate was adjusted on the basis of the methanol consumption rate. During fermentation, the broth was sampled periodically to determine the OD600, residual methanol, and L-lactate produced by using spectrometry and HPLC.

Adaptive laboratory evolution (ALE)
ALE was carried out in Delft-M10 in a 100 mL shake flask. The whole process is illustrated in Supplementary Fig. 8. Briefly, the parent strain HPLA06 was spotted on a YPD plate, and three independent colonies were cultivated in YPD medium at 37 °C and 220 r/min for 18 h. Strains were subsequently transferred to Delft-M10 medium at 37 °C and 220 r/min for 48 h, with an initial OD600 of 0.5. The evolved strains were transferred to the same medium with an initial OD600 of 0.5. After 8 transfers, the evolved strains with improved cell growth were subjected to L-lactate production from methanol.

Analytical methods
Cell growth was determined by measuring the optical density at 600 nm (OD600) via ultraviolet spectrophotometry. The fermentation sample was centrifuged at 13,000 × g for 5 min, and the supernatant was collected and filtered at 0.22 μm for determination. The concentrations of methanol and L-lactate were analyzed via HPLC (Shimadzu LC-2030, Japan) with an Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad, Hercules) equipped with an RID system. H2SO4 (5 mmol/L) was used as the mobile phase, with the column temperature and flow rate set to 50 °C and 0.5 mL/min, respectively. The results are presented as the means ± standard deviations (SDs) of three biological replicates.

Mass spectrometry (MS) analysis of lactate was carried out via HPLC–MS (Agilent 6540 Q-TOF). The HPLC procedure was the same as that used for HPLC detection, and it was conducted for 30 min. The MS parameters were as follows: ESI+/ESI- drying gas flow rate: 8 L/min; gas temperature: 350 °C; nebulizer gas pressure: 35 psi; capillary voltage: 3500 V; fragmentor voltage: 75 V; skimmer voltage: 65 V; octopole voltage: 750 V; and mass range: 50–1000. The mass-to-charge (m/z) peak at 89.0258 was attributed to C3H6O3 (lactate) without H atoms. The total number of samples was 3, and the number of controls (wild-type strain without expression of LDH gene) was 1. The fermentation sample was centrifuged at 13,000 g for 3 min, and then filtered through a 0.22 μm membrane. Then the sample was subjected to LC-MS detection. The software used in LC-MS was OpenLab ChemStation.

The identification of the optical isomer (D/L) of lactate was carried out via HPLC equipped with a UV detector by using a CHIRALPAK MA (+) column (DAICEL). CuSO4 (2 mM) was used as the mobile phase, and the column temperature and flow rate were set to 15 °C and 0.35 mL/min, respectively.

For cofactor ratio determination, the cells were collected at the mid-log phase (36 h for methanol cultivation and 18 h for glucose cultivation) and washed with phosphate-buffered saline (PBS) twice. The ratios of NADH/NAD+ and NADPH/NADP+ were measured according to our previous report12.

For promoter characterization, the eGFP fluorescence intensities of the engineered strains were detected as follows: 100 μL of cells cultured at different time points (2, 3, 4, and 5 d) were harvested and centrifuged at 3000 × g for 5 min. Then, the cell pellets were washed with 1 mL of H2O and diluted to OD600 = 0.2–0.8. The cell suspensions were subjected to a SPARK microplate reader (Tecan, Switzerland) in a 96-well black-walled clear-bottom plate for fluorescence determination, with excitation and emission wavelengths of 485 nm and 528 nm, respectively. All the fluorescence intensities were normalized to the OD600 (absorbance at 600 nm).

Detection of the cell death ratio by using PI staining in flow cytometry was carried out according to our previous study12. Simply put, pre-cultured strains were transferred to YPM medium with an initial OD600 = 0.4. After 24 and 48 h cultivation at 37 °C, 220 r.p.m., cells were collected by centrifugation at 1000 g for 5 min, then washed twice with pre-cold PBS. The supernatant was discarded, and the cells were resuspended in PI (5 μg/mL in PBS) for 10 min. Detection of PI fluorescence in each sample was analyzed on a flow cytometer (Sony) using FL-3 (488/620 nm). Then, 10,0000 cells were counted per sample to calculate the ratio of dead cells. Cells without PI staining were adopted to define the negative boundary. In the subsequent experiments, the same boundary and gating strategy was copied to calculate PI-positive cells.

Fluorescence microscopy analysis
To confirm mitochondrial targeting, MTS (MLSLRQSIRFFKPATRTLCSSRYLLQ) was fused to the N-terminus of the green fluorescent protein eGFP, and the samples were transformed into the yeast strain k–L. The engineered strain was incubated at 220 rpm at 30 °C in YPD medium for 24 h. A total of 1 mL of cell culture was sampled and centrifuged at 3000 × g for 5 min. Then, the cell pellets were suspended in Mito-Tracker Red Chloromethyl-X-rosamine (CMXRos) solution (Mito-Tracker Red CMXRos: PBS = 1:1000) preheated at 37 °C and incubated at 37 °C for 30 min. The incubated cells were centrifuged at 3000 × g for 5 min, and the supernatant was resuspended in preheated PBS buffer at 37 °C. Afterward, 1 μL of cell culture was dropped onto microscope slides and covered with cover glass, and then, fluorescence targeting was observed with an EVOS M5000 microscope (Thermo Fisher Scientific).

TEA and LCA analysis
Process simulation. A schematic depicting the generation of L-lactate from a CO2-derived methanol process is shown in Fig. 6a. This study entailed the design of an L-lactate production process using Aspen Plus v14 software, which was utilized to conduct calculations for mass and energy balance. The phase equilibrium and thermodynamic model has been constructed using the NRTL (non-random two-liquid), in accordance with a previous study70. The L-lactate manufacturing process consisted of five components: methanol production (A100), L-lactate production (A200), L-lactate purification (A300), wastewater treatment (A400), and utility (A500). The principal assumptions of Aspen Plus v14 are presented for M10 in a 100 mL shake flask. The assumptions of the whole process are illustrated in Supplementary Table 2. The detailed process simulation is described in the Supplementary Information.

Techno-economic analysis. TEA is a comprehensive methodology utilized to evaluate the feasibility of L-lactate production from CO2-derived methanol, considering both economic and technical perspectives. The estimation of CAPEX, OPEX, and MSP was conducted via an in-house model created by the National Renewable Energy Laboratory (NREL). Figure 6a shows the mass balance of the L-lactate production process simulated in Aspen Plus, with comprehensive mass and energy balance data available in Supplementary Data 4–7 in Source Data and Tables 11–13. The inside battery limit (ISBL) is typically utilized in TEA for fundamental considerations related to warehouse investments, site development, and supplementary piping. This study indicates that the ISBL encompasses the costs associated with equipment and installation for A100 to A300 (Fig. 6a). The fundamental assumptions and essential parameters for the process scale-up are presented in Supplementary Table 3 and are derived from reports and studies conducted by the NREL.

The biorefinery is projected to have an annual L-lactate production capacity of 18,500 tons, considering various influencing factors, including equipment size, raw material costs, and other related expenses. The costs associated with the equipment and raw materials utilized in the estimation of CAPEX and OPEX were sourced from published studies and official reports (Supplementary Table 13). The levelized cost of electricity for utility-scale photovoltaic solar power in the base scenario is $0.031 KWh−1, based on the latest data for high-efficiency regions in 202471. The costs associated with equipment varied according to the size of implementation and followed a power law relative to the original price72. The TEA functioned as an nth-plant model capable of estimating the investment required for the precommercial process72. The discounted cash flow method allowed for the determination of the MSP of L-lactate at the juncture where the NPV was zero and the IRR was 10%. All costs were adjusted to reflect 2024 US dollars to mitigate the impact of inflation on expenses34,72.

Life cycle assessment. The aim of the LCA was to quantify and analyze the environmental impacts of producing L-lactate from CO2. The system boundary was from cradle to gate (Fig. 6e). All stages, from raw material generation to L-lactate synthesis, were included inside the system boundaries. One kilogram of product mass was generally regarded as the functional unit in chemical LCA studies; hence, we utilized 1 kg of L-lactate as the functional unit to facilitate comparison with other research employing the same methods, thereby establishing a standard for both inputs and outputs. We investigated the L-lactate production route utilizing raw ingredients and energy inputs obtained from Aspen Plus. Supplementary Table 7 provides a comprehensive inventory of the life cycle associated with L-lactate production, derived from Aspen Plus. Moreover, we assessed the GWP (kg CO2-eq) of the L-lactate production process. The life cycle evaluation included background data from Ecoinvent v3.6 and references73, encompassing detailed categories and providers (Supplementary Data 8 in Source Data). The tool for reduction and assessment of chemicals and other environmental impacts (TRACI) version 2.1, created by the Environmental Protection Agency (EPA), was utilized to provide characterization elements for evaluating the possible consequences of inputs and emissions from production processes. The economic allocation method is chosen to assess the GWP of the by-product (biofertilizer)74. The cost of L-lactate is believed to correspond with the MSP of the basic scenario from this study, while Supplementary Table 13 encompasses the price of biofertilizer. All calculations and estimations are predicated on openLCA software version 2.2.

Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript “Methanol biotransformation for the production of the biodegradable plastic monomer L-lactate in yeast” by Yu et al. is well-written and easy to follow. This study explores L-lactic acid production in the methylotrophic yeast Ogataea polymorpha using methanol as a substrate. The authors demonstrate that balancing the NADH/NAD⁺ and NADPH/NADP⁺ cofactor ratio significantly enhances L-lactate synthesis. Additional metabolic engineering strategies included mitochondrial compartmentalization of LDH and improving cell viability by deleting IZH3. Adaptive laboratory evolution (ALE) experiments improved strain robustness toward methanol but did not enhance lactic acid production. In a fed-batch fermentation setup using CO₂-derived methanol, the engineered strain produced 25 g/L L-lactate. A key strength of this study is the combination of genetic engineering approaches—such as optimizing reductive power and compartmentalization—to improve lactic acid production from methanol. Furthermore, the authors integrate their experimental data into techno-economic analysis (TEA) and life cycle assessment (LCA), providing valuable industrial context for future applications.\nI suggest the following points for improvement:\n• Lines 101-102: The authors tested five Ldh enzymes in O. polymorpha, with two (Lactobacillus delbrueckii and Lactobacillus helveticus) being non-functional. Could the authors clarify why these enzymes did not work in O. polymorpha? Gene expression analysis of all five Ldh enzymes would help confirm whether they were actively expressed.\n• Line 31: \"Highest titer\" is more appropriate than \"yield\" in this context.\n• Line 120: The manuscript states that HPLA06’s growth decreased by 32.6% compared to the wild-type strain and refers to two separate figures (Fig. 2b and 2d). Presenting this phenotype in a single diagram—rather than requiring readers to compare two figures—would improve clarity. Consider adding a consolidated figure to the Supplementary Information section.\n• Lines 154-155: It would be helpful to specify that pGAP-driven LDH constructs were used for this analysis. Otherwise, readers might assume that the previously mentioned pAOX1 strains were cultivated under glucose conditions.\n• Discussion: One of the manuscript’s key strengths is incorporating TEA and LCA to contextualize the experimental findings. However, this aspect is not sufficiently discussed. Adding a paragraph on TEA/LCA in the discussion section would strengthen the manuscript.\nSupplementary Information (SI) Suggestions:\n• SI Appendix Figure S1 (a) and (b) should be explicitly mentioned in the figure description.\n• SI Appendix Figure S11: Ensure a consistent font size in the figure description.\n• SI Appendix Figures S4 and S9: The phrase in situ does not need to be italicized, as it is commonly used in this research field. Please check for consistency throughout the manuscript.", "reviewer_4": "This manuscript describes the engineering of the methylotrophic yeast O. polymorpha for efficient production of L-lactate from CO2-derived methanol, integrating systematic metabolic engineering with comprehensive techno-economic and life cycle assessments. The study is notable not only for successfully developing a methanol-utilizing yeast strain with reasonably high lactate production,but also for providing a pioneering, systematic evaluation of the economic and environmental impacts associated with this CO2-to-chemical scenario. However, several issues need to be addressed before the manuscript can be recommended for publication:\n1. Although yeasts generally tolerate acidic conditions, the lactate tolerance limit of O. polymorpha should be studied and discussed.\n2. Lines 127-128: The authors observed improved cell viability upon disruption of IZH3 but not LPL1. Please discuss the potential mechanisms underlying the differential effects of these disruptions.\n3. Lines 142-143: The authors suggest that NADPH is more abundant in methanol metabolism compared to glucose metabolism, which is very interesting. However, the stated “methanol assimilation via the pentose phosphate pathway (PPP) and the Xu5P cycle” seems to imply a metabolic cycle predominantly generating NADPH while releasing CO2, which suggests limited or no net C1 assimilation. Please provide a detailed explanation to clarify this aspect.\n4. Lines 152-153: The NAD(P)H/NAD(P)+ ratio can significantly fluctuate under different cultivation conditions. Given that this ratio was measured during the log growth phase, please clarify whether the high NADPH/NADP+ ratio observed also applies during later stages of production.\n5. The term \"cofactor balance\" should be used cautiously. It may be valid to claim improved cofactor balance in strain HPLA13 relative to HPLA06; however, it seems questionable to state that HPLA13 outperforms the WT strain in terms of cofactor balance, since the WT strain inherently maintains cofactor balance through evolution.\n6. Lines 182-183: How was the \"CO2-derived methanol solution\" produced to compose methanol and water at a precise 3:1 ratio? Could the authors clarify how this exact ratio was achieved and confirm whether the phrase \"without byproduct\" specifically means \"without impurities\"?\n7. Lines 201-202: When discussing the economic contributions of individual modules, please include specific percentage values.\n8. For TEA and LCA, can the reuse of residual biomass (e.g., for single-cell protein, SCP) be considered to further valorize the CO2-to-lactate manufacturing process?\n9. Lines 286-289: The authors should be cautious in drawing conclusions about the engineered strain's ALE performance without explicitly examining genetic stability.", "reviewer_5": "Biodegradable plastics such as polylactic acid (PLA) are gaining attention as sustainable alternatives to fossil-derived polymers with emerging C1-fuelled bioprocesses offering a promising route for efficient and eco-friendly lactate production. In this study, O. polymorpha was metabolically engineered to significantly increase the L-lactate production yield using methanol alone. The authors propose a promising strategy to combine liquid sunshine technology with biotransformation of methanol using metabolically engineered O. polymorpha yeast to increase L-lactate yield and evaluate its economic feasibility and GHG mitigation performance.\nHowever, it is unable to decide to be published in Nature Communications for some major and minor points while It has no doubt that the author’s research will be of interest to fellow specialists working in the field of metabolic engineering. There are serious concerns about assessment methods and results that have a significant impact on the clarity of the paper.\nMajor points\n1) In Table S12, the reference paper presenting the cost of solar power provides a cost of $0.05/kWh for solar power in 2017. However, the source of the cost remains unclear as it is uncertain whether it originates from photovoltaics or not. A range of statistics indicates that the price of electricity from the grid varies between $0.08 and $0.25 per kWh. As stated in the NREL report (Kazi et al., National Renewable Energy Lab (NREL), 2010), the electricity price was assumed to be $0.054/kWh in 2007. In consideration of these reports, the cost of solar power in this study cannot be considered a viable cost. Since the cost of solar power shows a high sensitivity in the techno-economic analysis, further explanation for the decrease of the cost of solar power from 2017 to 2024 and a reference to the cost of solar derived power in 2024 are needed.\n2) The mass and energy balance and specific estimation of capital expenditures (CapEx) of each equipment from A100 to A500 should be presented in the supporting information file, respectively. Table S6 mentions the amounts of the primary materials but the mass and energy balance for each subsystem is not available. Table S4 mentions capital expenditures for each area, but more detailed information for each equipment is not available too.\n3) The biofertilizer produced by anaerobic digestion of the biomass is considered economic credit in Table S12, but not included in the environmental impact assessment in Table S6-S7. It is crucial to address the different methodology between the economic assessment, which incorporated biofertilizer credits, and the environmental assessment, which made no consideration.\n4) It is necessary to specify the impact assessment methodology such as ReCiPe 2016 Midpoint (H), Environmental Footprint 3.1 and TRACI 2.1 on which the life cycle assessment is based. In Table S6, the specified life cycle inventory data for the source of the material and energy are also required. It should include the information where the data origins from (GLO, RER, RoW, ⋯) and whether the data is cut-off (APOS, Cut-off, ⋯)\n5) A detailed description of the thermodynamic model selected to simulate the process is required.\nMinor points\n1) The manuscript should provide a reason to support the assumption of an L-lactate production capacity of 18,500 tons/year.\n2) It is recommended to improve the visibility of the text and numbers in Figure 6 to enhance overall clarity.\n3) There is an inconsistency in the use of the terms ‘lactate’ and ‘lactic acid’ between the main text and the supporting information file.\n4) In table S4, there is a typo. ‘Total operating cost’ should be corrected to ‘Total capital cost’\n5) In table S5, which presents the operating expenditures, the use of the term ‘investment’ should be appropriately revised.", "reviewer_6": "The present manuscript combined three diverse areas in a single paper, lacking thorough justice to individual topics, especially TEA. The TEA used a completely wrong approach for process design, which makes the results unreliable and unacceptable, I recommend removing TEA work from the manuscript to avoid passing the wrong information to the readers.\nBelow are my few comments.\n•Supporting Information: The reaction stoichiometry for methanol production (2 CO2 + 4 H2 → 2 CH3OH + O2) seems to be vague. Also, there are no references to support the reaction conditions for methanol production. Also, I could not find the oxygen as a byproduct.\n•What is the basis of plant capacity?\n•TEA: There is no mention of low pH or neutral fermentation.\n•L-Lactate is mentioned in the manuscript. However, the reaction stoichiometry shows L-lactic acid.\n•L-lactate purification (A300): It looks to be completely vague. It may be noted that LA separation is the major cost contributor to the overall process in terms of capital investment and energy consumption. If this kind of downstream separation is considered, the TEA results become completely unreliable. Please refer to recent papers on LA production. Otherwise, I recommend removing TEA completely from the manuscript as it will pass wrong information to the readers.\n•AD: Reaction stoichiometry and conditions are not mentioned.\n•The complete Aspen Plus flowsheet is missing. The details of the equipment and their design parameters are also missing. As a result, it is difficult to judge the reliability of TEA results.\n•The energy cost is completely missing from TEA, which is one of the main cost contributors in this process.\n•“The estimation of CAPEX, OPEX, and MSP was conducted via an in-house model created by the National Renewable Energy Laboratory (NREL).” Is it an open-source software? Where is the link?", "reviewer_7": "Please see the attached file"}, "author_response": ""}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all questions and comments satisfactorily. I am fine with the current version and support its publication. There are just a few minor writing issues that should be corrected.\nMinor corrections\nLine 111: in situ should not be italicized.\nLine 160: Please be mindful of the tense in this sentence (\"It is worth mentioning...\").", "reviewer_4": "After careful review, the authors have satisfactorily addressed all questions raised by the reviewers. The manuscript is now suitable for publication in Nature Communications."}, "author_response": "Responses to the reviewers’ comments (NCOMMS-25-14369)\nReviewer #5:\nGeneral comment\nBiodegradable polymers like polylactic acid (PLA) are increasingly recognized as eco‑friendly substitutes for petroleum-based plastics, and novel single‑carbon (C1) bioprocesses present an efficient pathway for lactate synthesis. The authors introduce an integrated approach that combines “liquid sunshine” photochemical conversion with engineered O. polymorpha fermentation to enhance L-lactate titers, and assess the system’s techno-economic viability alongside its greenhouse-gas reduction potential.\nThe assessment methodologies have been appropriately addressed in the revised manuscript and supplementary file with a clear explanation, and the results are clearly presented and discussed in the context of relevant literature. The authors have addressed all the major and minor comments appropriately, and I think this paper can be published in Nature Communications now.\nDecision: Accept\nMajor points\nComment 1. Thank you for the revision and efforts to address the comment. The authors explained that the levelized cost of electricity for utility-scale photovoltaic solar power in 2024 is approximately $0.031-0.075/kWh with references. However, some references, such as the Fraunhofer report (2024), present the levelized cost of electricity for photovoltaic solar power as a range of $0.04-0.24/kWh. It is recommended to consider the various ranges of the levelized cost of photovoltaic solar power, rather than focusing solely on the minimum cost. Considering that photovoltaic solar power is a form of renewable energy, its price varies greatly depending on the region, making it infeasible to reflect the minimum price in a specific region. The authors should consider the price of photovoltaic solar power in various regions and perform the techno-economic analysis for two cases: when the solar power cost is minimum and when it is maximum. The Fraunhofer report (2024): Christoph Kost et al., Levelized Cost of Electricity Renewable Energy Technologies, Fraunhofer, 2024.\nReply 1: Thank you for your detailed comment. In the revised manuscript, we incorporated sensitivity analysis scenarios by using utility-scale photovoltaic power prices from different regions, drawing on multiple references including Fraunhofer (2025)1-6. A corresponding sensitivity curve is provided in Supplementary Fig. S15. We also explicitly discussed the regional variations in photovoltaic electricity prices in the manuscript (page 10, lines 248-255):\n“Considering the regional variation in solar power costs, the MSP fluctuates significantly with the geographical location of the plant43 (Supplementary Fig. S15). For example, countries with abundant solar resources such as Pakistan show solar power prices close to the global minimum (<0.025 $/kWh), resulting in comparatively lower MSP, whereas regions with higher solar power prices such as Canada (>0.10 $/kWh) may increase the MSP by over 13%. These results emphasize the critical role of site selection in improving the economic competitiveness of CO -to-L-lactate 2 biomanufacturing.”\nSupplementary Fig. S15 Regional impact of utility-scale photovoltaic solar levelized cost of electricity (LCOE) on the minimum selling price of L-lactate in 2024. Data sources: Pakistan1, India2, global average2, United States3, Southern Germany4, China5, Northern Germany4, Canada6.\nReviewer’s response:\nThis comment has been appropriately addressed in the revised manuscript with a clear explanation.\nResponses to the reviewers’ comments (NCOMMS-25-14369)\nDear reviewers,\nWe would like to express our thanks to the constructive suggestions regarding to our manuscript “Methanol biotransformation for the production of the biodegradable plastic monomer L-lactate in yeast”. These comments are helpful for us to revise and further improve our paper. The main corrections and responses to the reviewers’ comments are as follows:\nResponses to Reviewer 1\nGeneral comment: The manuscript “Methanol biotransformation for the production of the biodegradable plastic monomer L-lactate in yeast” by Yu et al. is well-written and easy to follow. This study explores L-lactic acid production in the methylotrophic yeast Ogataea polymorpha using methanol as a substrate. The authors demonstrate that balancing the NADH/NAD⁺ and NADPH/NADP⁺ cofactor ratio significantly enhances L-lactate synthesis. Additional metabolic engineering strategies included mitochondrial compartmentalization of LDH and improving cell viability by deleting IZH3. Adaptive laboratory evolution (ALE) experiments improved strain robustness toward methanol but did not enhance lactic acid production. In a fed-batch fermentation setup using CO₂-derived methanol, the engineered strain produced 25 g/L L-lactate. A key strength of this study is the combination of genetic engineering approaches—such as optimizing reductive power and compartmentalization—to improve lactic acid production from methanol. Furthermore, the authors integrate their experimental data into techno-economic analysis (TEA) and life cycle assessment (LCA), providing valuable industrial context for future applications.\nReply: We appreciate your positive comments on our work. Here, we provide a point-by-point reply on all your comments, which we hope to address your concerns.\nComment 1: Lines 101-102: Could the authors clarify why these enzymes did not work in O. polymorpha? Gene expression analysis of all five Ldh enzymes would help confirm whether they were actively expressed.\nReply 1: Thanks for your constructive comments. As suggested, qPCR was carried out in engineered strains with these five Ldh enzymes. As shown in Fig. R1-1B, all five LDH genes were functionally expressed at the transcriptional level. Particularly, strains failing to produce LA (HpLd and HpLh) showed higher LDH transcriptional levels, probably owing to the better growth behavior at the sampling points. Therefore, no LA detected in strain HpLd and HpLh could be attributed to that these two Ldh enzymes did not work in O. polymorpha, which is commonly observed in heterologous expression (Bioresour. Technol., 2025, 415, 131730). Related information was also involved in the revised main text (Line 101-102) and Fig S3a.\nA B\n1200 7 10\nle )L /g m ( e ta tc a L 10 4 6 8 0 0 0 00 0 0 0 2 3 4 5 6 D O 0 0 6 v e l n o is s e rp x e e v 4 6 8 L- 200 1 ita le 2 0 0 0 Δku80 HpLd HpLh HpLp HpLl HpPa LΔdkLu8D0HLhHLpLDdHLHppLLDhHLHlLpDLpHPaHLpDLlHHpPa LDH Ld Lh Lp Ll Pa LDH Ld Lh Lp Ll Pa\nFigure R1-1 Screening functional Ldh enzymes in O. polymorpha.\nComment 2: Line 31: \"Highest titer\" is more appropriate than \"yield\" in this context.\nReply 2: Thank you for pointing out this detail. We have replaced “yield” with “titer” in Line 30.\nComment 3: Line 120: The manuscript states that HPLA06’s growth decreased by 32.6% compared to the wild-type strain and refers to two separate figures (Fig. 2b and 2d). Presenting this phenotype in a single diagram—rather than requiring readers to compare two figures—would improve clarity. Consider adding a consolidated figure to the Supplementary Information section.\nReply 3: Thank you for your valuable suggestion. We compared wild-type strain with HPLA05 and HPLA06 in a single experiment. Cell growth and LA were detected at different time points. Similar to the previous results, HPLA05 and HPLA06 with efficient LA production showed obviously decreased growth (Figure R1-2). We have added a consolidated figure in Supplementary Fig. S5, and replaced the figure annotation in the manuscript with Supplementary Fig. S5.\na b\n8 2\nControl (Δku80L) Control (Δku80L)\n1.6\nD\nO\n006\n3\n4\n5 /g (\ne ta\ntc a 0\n1\n.\n.\n8\n2\n0.4\n1\n0 0\n0 20 40 60 80 0 20 40 60 80\nTime (h) Time (h)\nFigure R1-2 Engineered strain HPLA05 and HpLA06 for L-lactate production, significantly decreased biomass relative to that of the wild-type strain.\nComment 4: Lines 154-155: It would be helpful to specify that pGAP-driven LDH constructs were used for this analysis. Otherwise, readers might assume that the previously mentioned pAOX1 strains were cultivated under glucose conditions.\nReply 4: Thank you for your valuable suggestion. To avoid the misunderstanding for readers, we have specified the use of pGAP-driven LDH constructs in glucose cultivation in Line 160-161.\nComment 5: Discussion: One of the manuscript’s key strengths is incorporating TEA and LCA to contextualize the experimental findings. However, this aspect is not sufficiently discussed. Adding a paragraph on TEA/LCA in the discussion section would strengthen the manuscript.\nReply 5: Thank you for your insightful suggestion regarding the insufficient discussion. We have added a dedicated paragraph in the discussion section to explore the implications and prospects of L-lactate production via the CO -derived methanol 2 pathway, in relation to our TEA and LCA findings. The key points are summarized below. A comprehensive revision can be found on Line 355-371:\n“The integration of TEA and LCA facilitated a comprehensive assessment of the CO -to-L-lactate 2 biomanufacturing process, revealing essential insights beyond basic experimental performance. Although the competitive potential is inadequate to that of established sugar-based and lignocellulosic processes (Supplementary Table S9), this study emphasize the importance of third- generation biomanufacturing — particularly regarding reduced carbon source costs, optimized process utility, and carbon credit considerations — which collectively improve economic viability, resulting in an MSP lower than the market price of L-lactate. Furthermore, it is imperative to develop the TEA to identify key factors affecting return on investment, thereby guiding experiments, and mitigating investment risks67. This research demonstrated that production characteristics, including yield and productivity, are key cost determinants, providing explicit targets for metabolic engineering and process intensification to reduce MSP and enhance scalability. The LCA demonstrated that utilizing CO -derived methanol as a carbon source facilitated a net GHG capture 2 of 7.29 tones CO per ton of product, establishing this pathway as both carbon-negative and 2 economically feasible. The TEA and LCA confirm the viability and sustainability of L-lactate production through CO -derived methanol and provide strategic direction for the future 2 advancement of carbon-based biomanufacturing.”\nComment 6: SI Appendix Figure S1 (a) and (b) should be explicitly mentioned in the figure description.\nReply 6: Thank you for your valuable suggestion. We have revised the figure description of Figure S1 and added explicit explanation of Fig. S1 (a) and (b).\nComment 7: SI Appendix Figure S11: Ensure a consistent font size in the figure description.\nReply 7: Thank you for your valuable feedback. We have carefully reviewed the figure description and ensured that all textual elements conform to a consistent font size standard. The amended version has been included in the Supplementary Information (now Supplementary Fig. S12).\nComment 8: SI Appendix Figures S4 and S9: The phrase in situ does not need to be italicized, as it is commonly used in this research field. Please check for consistency throughout the manuscript.\nReply 8: Thank you for pointing out this detail. We have checked and revised all the “in situ” throughout the manuscript and Supplemental Information.\nResponses to Reviewer 4\nGeneral comment: This manuscript describes the engineering of the methylotrophic yeast O. polymorpha for efficient production of L-lactate from CO -derived methanol, 2 integrating systematic metabolic engineering with comprehensive techno-economic and life cycle assessments. The study is notable not only for successfully developing a methanol-utilizing yeast strain with reasonably high lactate production, but also for providing a pioneering, systematic evaluation of the economic and environmental impacts associated with this CO -to-chemical scenario. However, several issues need 2 to be addressed before the manuscript can be recommended for publication:\nReply: We appreciate your encouraged comments on our manuscript, and we provide a point-by-point reply to address your concerns.\nComment 1: Although yeasts generally tolerate acidic conditions, the lactate tolerance limit of O. polymorpha should be studied and discussed.\nReply 1: Thank you for your valuable suggestion. We have conducted a tolerance experiment, where O. polymorpha wild type strain k-L was cultivated in various concentrations of L-lactate with an initial OD =0.2. The results showed that 5 g/L L- 600 lactate exhibited no obvious growth inhibition to O. polymorpha (Supplemental Fig. S6). In practical fermentation where L-lactate is produced and secreted to the medium gradually, especially in stationary phase, the tolerance may reach 10-fold (50 g/L). Just as James C. Liao et al. (Non-fermentative pathways for synthesis of branched-chain higher alcohols as biofuels, Nature, 2008, 451, 3) reported that the E. coli strain with tolerance of 15 g/L isobutanol can produce about 20 g/L isobuatnol in shake flask, which may reach 10-fold in bioreactor. We have added this description in Discussion in Line 349-354.\n16\n14\n12\n10\n0\nO\n6\n4\n2\n0\n0 1 2 5 10\nL-lactic acid (g/L)\nFigure R4-1 L-lactate tolerance of wild-type O. polymorpha.\nComment 2: Lines 127-128: The authors observed improved cell viability upon disruption of IZH3 but not LPL1. Please discuss the potential mechanisms underlying the differential effects of these disruptions.\nReply 2: Thank you for your valuable suggestion. In our previous study, we found that mutation of IZH3 and LPL1 was responsible for growth recovery of fatty acid producing strain which cannot grow in methanol. LPL1 possesses the activity of phospholipase/lysophospholipase for degrading various phospholipid substrates and IZH3 is a membrane protein related to zinc metabolism. izh3Δ mutation was found to alleviate the DNA damage caused by antimicrobial peptide (Molecular Microbiology, 2007, 65: 494-507), and it is speculated that the izh3Δ mutation may also attenuate the damaging effects of formaldehyde during L-LA production. In contrast, the disruption of LPL1 blocked phospholipid degradation, which may direct more carbon flux from pyruvate to acetyl-CoA to synthesize lipid, therefore the flux from pyruvate to L-lactate was reduced. We have added this description to the Discussion in Line 320-329.\nComment 3: Lines 142-143: The authors suggest that NADPH is more abundant in methanol metabolism compared to glucose metabolism, which is very interesting. However, the stated “methanol assimilation via the pentose phosphate pathway (PPP) and the Xu5P cycle” seems to imply a metabolic cycle predominantly generating NADPH while releasing CO , which suggests limited or no net C1 assimilation. Please 2 provide a detailed explanation to clarify this aspect.\nReply 3: Thank you for your advice. NADPH generated from the pentose phosphate pathway that release CO₂, which is a fact in both methanol and glucose metabolism. And Xu5P is necessary for formaldehyde assimilation.\nComment 4: Lines 152-153: The NAD(P)H/NAD(P)+ ratio can significantly fluctuate under different cultivation conditions. Given that this ratio was measured during the log growth phase, please clarify whether the high NADPH/NADP+ ratio observed also applies during later stages of production.\nReply 4: Thanks for your reasonable suggestion. As detailed in the manuscript, the cofactor ratios were measured during the log growth phase (~36 h), when lactate dehydrogenase (Ldh) activity peaked. Specifically, time-course analysis of growth and lactate (LA) production (Figure R4-2) revealed that LA biosynthesis was primarily confined to the log phase. This temporal correlation directly determined our focus on cofactor ratios during this critical period. It is true that cofactor may exhibit variability under different cultivation parameters (e.g., stationary phase), such fluctuations do not appear to impact the LA production under our experimental conditions.\n3.5\nSampling at 36 h D O\n0063.0 HpLA06\nOD 600\n&\n2.5\n4.0\nNADH/NAD+ )L 2.0\n/g\no ita\nr\n3.0 NADPH/NADP+ ( e ta\ntc 1\n1\n.\n.\n0\nr o\tc\n2.0 a L-l 0.5\na 0.0\nfo\nC 1.0 3.0\n0 20 40\nTime (h)\n60 80 100\nHpLA13\n0.0 D\n006 2.5\nOD 600\nStrain k-L HPLA06 HPLA13 O 2.0\n&\nLdh －\nde\nN\np\nA\nen\nD\nd\nH\ne nt de\nN\np\nA\ne\nD\nn\nP\nde\nH\nn t\n)L\n/g\n1.5\nLA\n(\ne 1.0\ta\ntc 0.5\na\nL-l\n0.0\n0 20 40 60 80 100\nFigure R4-2 L-lactate was primarily produced during the log growth phase, when the cofactor ratios were analyzed.\nComment 5: The term \"cofactor balance\" should be used cautiously. It may be valid to claim improved cofactor balance in strain HPLA13 relative to HPLA06; however, it seems questionable to state that HPLA13 outperforms the WT strain in terms of cofactor balance, since the WT strain inherently maintains cofactor balance through evolution.\nReply 5: Thank you for your valuable suggestion. Cofactor balance is indeed not applicable to the WT strain, therefore we deleted the comparison of HPLA13 with WT strain.\nComment 6: Lines 182-183: How was the \"CO -derived methanol solution\" produced 2 to compose methanol and water at a precise 3:1 ratio? Could the authors clarify how this exact ratio was achieved and confirm whether the phrase \"without byproduct\" specifically means \"without impurities\"?\nReply 6: Thank you for pointing out this detail. The \"CO -derived methanol solution\" 2 was produced via CO hydrogenation by High-Performance MaZrOx (Ma = Cd, Ga) 2 Solid-Solution Catalysts, under the reaction conditions of H /CO = 3/1 (Wang et al. 2 2 ACS Catal, 2019, 9, 10253-10259). The reaction is 3H +CO =CH OH+H O, therefore 2 2 3 2 the molecular ratio of methanol and water is 1:1, and the mass ratio is 32:18. Therefore the v/v of methanol and water is 32/0.791:18/1=2.25:1. We are so sorry for making this mistake, and we have revised the ratio from 3:1 to 2.25:1 (v/v) in the manuscript (Line 188).\nAnd the phrase \"without byproduct\" specifically means \"without impurities\". That is to say, the solution contains nothing but methanol and water. We have revised this description in the manuscript (Line 189).\nComment 7: Lines 201-202: When discussing the economic contributions of individual modules, please include specific percentage values.\nReply 7: Thank you for your constructive feedback. We have added specific percentage values to clearly present the economic contributions of each module, covering both capital expenditures (Supplementary Table S4) and minimum selling price (Supplementary Table S6). These revisions are detailed in Line 209-213, and 226-227 of the revised manuscript:\n“Among the five modules of the process (methanol production, L-lactate production, L-lactate purification, wastewater treatment, and utilities), the L-lactate production subsystem was determined to necessitate the greatest installation cost for equipment. The CAPEX for equipment and installation of five modules were 2.3%, 59.5%, 12.3%, 7.3%, and 18.6%, respectively (Supplementary Table S4).”\n“When we assumed operation of the L-lactate production process for 30 years with an internal rate of return (IRR) of 10%, the net present value (NPV) became zero at an L-lactate minimum selling price (MSP) of $2.29 kg-1, which was comparable to the prevailing market price of $1.85-3.15 kg- 1 35, 42. The cost distribution for the MSP of five modules was 31.2%, 43.9%, 9.5%, 3.0%, and 12.4%, respectively (Supplementary Table S6).”\nComment 8: For TEA and LCA, can the reuse of residual biomass (e.g., for single-cell protein, SCP) be considered to further valorize the CO2-to-lactate manufacturing process?\nReply 8: Thank you for your insightful inquiry. We have evaluated the valorization of residual biomass in our TEA and LCA. In our research, residual biomass is employed through anaerobic digestion, and the solid digestate is sold as biofertilizer1, 2. The approach yields economic and environmental advantages, producing an anticipated annual profit of around $2.14 million (Supplementary Table S5) and allowing for a reduction of 0.277 kg CO -eq from the total GWP of the process (Supplementary Table 2 8). Detailed revision can be found in Line 222-223 and 263-265:\n“Furthermore, the residual biomass can be marketed as nitrogen fertilizer through anaerobic digestion.”\n“It is worth mentioning that from the standpoint of economic value allocation, the by-product of fertilizer could result in a GWP decrease of 0.277 kg CO2-eq, so further diminishing the carbon footprint.”\nReferences:\n1. Zhang, C. et al. An upcycling bioprocess for sustainable aviation fuel production from food waste-derived greenhouse gases: Life cycle assessment and techno-economic analysis. Chemical Engineering Journal 486, 150242, 150242 (2024).\n2. Yan, X. et al. Paradigm of engineering recalcitrant non-model microorganism with dominant metabolic pathway as a biorefinery chassis. Nature Communications 15, 10441 (2024).\nComment 9: Lines 286-289: The authors should be cautious in drawing conclusions about the engineered strain's ALE performance without explicitly examining genetic stability.\nReply 9: Thank you for the valuable suggestion. Actually, ALE decreased L-lactate production, but the mechanism was still unknown, especially the genome stability was not explored. Therefore, to be cautious, we deleted the conclusion about trade-off between lactate production and biomass accumulation. We just stated that fact that ALE decreased L-lactate production from methanol, and the mechanism is worth being explored in the future (Line 138-139).\nResponses to Reviewer 5\nGeneral comment: Biodegradable polymers like polylactic acid (PLA) are gaining attention as sustainable alternatives to fossil-derived polymers with emerging C1- fuelled bioprocesses offering a promising route for efficient and eco-friendly lactate production. In this study, O. polymorpha was metabolically engineered to significantly increase the L-lactate production yield using methanol alone. The authors propose a promising strategy to combine liquid sunshine technology with biotransformation of methanol using metabolically engineered O. polymorpha yeast to increase L-lactate yield and evaluate its economic feasibility and GHG mitigation performance.\nHowever, it is unable to decide to be published in Nature Communications for some major and minor points while it has no doubt that the author’s research will be of interest to fellow specialists working in the field of metabolic engineering. There are serious concerns about assessment methods and results that have a significant impact on the clarity of the paper.\nReply: We appreciate your positive evaluation on our work. Here, we provide a point-by-point reply on all your comments, especially to clarify assessment methods and results of TEA and LCA.\nMajor points:\nComment 1: In Table S12, the reference paper presenting the cost of solar power provides a cost of $0.05/kWh for solar power in 2017. However, the source of the cost remains unclear as it is uncertain whether it originates from photovoltaics or not. A range of statistics indicates that the price of electricity from the grid varies between $0.08 and $0.25 per kWh. As stated in the NREL report (Kazi et al., National Renewable Energy Lab (NREL), 2010), the electricity price was assumed to be $0.054/kWh in 2007. In consideration of these reports, the cost of solar power in this study cannot be considered a viable cost. Since the cost of solar power shows a high sensitivity in the techno-economic analysis, further explanation for the decrease of the cost of solar power from 2017 to 2024 and a reference to the cost of solar derived power in 2024 are needed.\nReply 1: Thank you for your detailed feedback. We have updated the cost of solar power to reflect the most recent data available for the high-efficiency regions (e.g., United States, Europe, and China) in 2024. According to the latest reports, the levelized cost of electricity for utility-scale photovoltaic solar power in 2024 is approximately $0.031-0.075 /kWh3, 4. This updated value has been incorporated into our analysis and is now cited with an appropriate reference in Supplementary Table S21. The results of operating expenditures, minimum selling price, and sensitivity analysis have been revised in the Supplementary Tables S5-S6, Fig. 6c, and Fig. 6d. The updates are detailed in Line 503-504:\n“The levelized cost of electricity for utility-scale photovoltaic solar power in the base scenario is $0.031 KWh-1, based on the latest data for high-efficiency regions in 202470.”\nReferences:\n3. Chong, S., Wu, J. & Chang, I. S. Cost accounting and economic competitiveness evaluation of photovoltaic power generation in China-based on the system levelized cost of electricity. Renewable Energy 222, 119940, 119940 (2024).\n4. SolarPACES. Qinghai sets CSP tariffs at 0.55 yuan/kWh (7.5 cents/kWh), (2025).\nComment 2: The mass and energy balance and specific estimation of capital expenditures (CapEx) of each equipment from A100 to A500 should be presented in the supporting information file, respectively. Table S6 mentions the amounts of the primary materials but the mass and energy balance for each subsystem is not available. Table S4 mentions capital expenditures for each area, but more detailed information for each equipment is not available too.\nReply 2: Thank you for your constructive comments. We have incorporated the mass and energy balance from A100 to A500, obtained from Aspen Plus, offering an additional and transparent overview on the material and energy flows inside our system (Supplementary Tables S14-S20). Additionally, we have provided a comprehensive breakdown of the equipment and installation costs for each individual item, as depicted in Supplementary Fig. S13, to complement the area-level data previously shown in Supplementary Tables S4, which aligns with existing literature1. The detailed revisions can be found in Line 212-213 and 491-493:\n“Comprehensive investment details for equipment and installation corresponding to each individual component are available in Supplementary Fig. S14. Notably, the bioreactors accounted for the predominant share of expenditures associated with equipment and installation in CAPEX.”\n“Fig. 6a shows the mass balance of the L-lactate production process simulated in Aspen Plus, with comprehensive mass and energy balance data available in Supplementary Table S14-S20.”\nReferences:\n1. Zhang, C. et al. An upcycling bioprocess for sustainable aviation fuel production from food waste-derived greenhouse gases: Life cycle assessment and techno-economic analysis. Chemical Engineering Journal 486, 150242, 150242 (2024).\nComment 3: The biofertilizer produced by anaerobic digestion of the biomass is considered economic credit in Table S12, but not included in the environmental impact assessment in Table S6-S7. It is crucial to address the different methodology between the economic assessment, which incorporated biofertilizer credits, and the environmental assessment, which made no consideration.\nReply 3: Thank you for your valuable comment. To ensure consistency with the system boundaries used in the techno-economic analysis (TEA), we have now included the environmental credits from biofertilizer in the life cycle assessment (LCA). These credits were calculated using the economic allocation method5, aligning the environmental assessment with the approach taken in the economic analysis. Based on the economic allocation method, the biofertilizer permits a deduction of 0.277 kg CO - 2 eq from the overall GWP of the process. The revised LCA results are displayed in Fig. 6e-6f and Supplementary Table S8. Comprehensive updates of results and methodology are included in Line 263-265 and 530-533:\n“It is worth mentioning that from the standpoint of economic value allocation, the by-product of fertilizer could result in a GWP decrease of 0.277 kg CO -eq, so further diminishing the carbon 2 footprint.”\n“The economic allocation method is chosen to assess the GWP of the by-product (biofertilizer)73. The cost of L-lactate is believed to correspond with the MSP of the basic scenario from this study, while Supplementary Table S21 encompasses the price of biofertilizer.”\nReferences:\n5. Ijassi, W., Ben Rejeb, H. & Zwolinski, P. Environmental impact evaluation of co-products: decision-aid tool for allocation in LCA. The International Journal of Life Cycle Assessment 26, 2199-2214 (2021).\nComment 5: It is necessary to specify the impact assessment methodology such as ReCiPe 2016 Midpoint (H), Environmental Footprint 3.1 and TRACI 2.1 on which the life cycle assessment is based. In Table S6, the specified life cycle inventory data for the source of the material and energy are also required. It should include the information where the data origins from (GLO, RER, RoW, ⋯) and whether the data is cut-off (APOS, Cut-off, ⋯)\nReply 5: Thank you for your valuable suggestions. We have specified the impact assessment methodology used in the LCA as the TRACI version 2.1. Besides, the life cycle inventory data are derived from our Aspen Plus models, with comprehensive mass and energy data available in Supplementary Tables S14-S20. We also added the information where the data origins from and whether the data is cut-off into Supplementary Table S22, which details categories and providers from the Ecoinvent v3.6 database and corresponding references. Specific information regarding this methodology has been incorporated into the LCA methodologies section of the manuscript in Line 519-527.\n“Supplementary Table S7 provides a comprehensive inventory of the life cycle associated with L-lactate production, derived from Aspen Plus. Moreover, we assessed the GWP (kg CO -eq) of the 2 L-lactate production process. The life cycle evaluation included background data from Ecoinvent v3.6 and references72, encompassing detailed categories and providers (Supplementary Table S22). The tool for reduction and assessment of chemicals and other environmental impacts (TRACI) version 2.1, created by the environmental protection agency (EPA), was utilized to provide characterization elements for evaluating the possible consequences of inputs and emissions from production processes.”\nComment 5: A detailed description of the thermodynamic model selected to simulate the process is required.\nReply 5: Thank you for your suggestion. The NRTL (non-random two-liquid) model was chosen to characterize the thermodynamic behavior of the system in our research. This model is widely recognized for its accuracy in predicting liquid-phase activity coefficients and phase equilibria in non-ideal mixtures, particularly those involving polar compounds and hydrogen bonding—key characteristics of our process (e.g., L-lactate fermentation broths, methanol-water systems)6. This model was implemented in Aspen Plus, and its suitability for our process is further supported by prior studies on biomanufacturing7-9. A detailed description of the thermodynamic model selection can be found in Line 478-482.\n“This study entailed the design of an L-lactate production process using Aspen Plus v14 software, which was utilized to conduct calculations for mass and energy balance. The phase equilibrium and thermodynamic model has been constructed using the NRTL (non-random two-liquid), in accordance with a previous study69.”\nReferences:\n6. Haydary, J. Chemical process design and simulation: Aspen Plus and Aspen Hysys applications. (John Wiley & Sons, 2019).\n7. Méndez-Alva, J. A. et al. in Computer Aided Chemical Engineering Vol. 44 463-468 (Elsevier, 2018).\n8. Gasca-González, R. et al. in Computer Aided Chemical Engineering Vol. 46 (eds Anton A. Kiss, Edwin Zondervan, Richard Lakerveld, & Leyla Özkan) 457-462 (Elsevier, 2019).\n9. Kruschitz, A. & Nidetzky, B. Biocatalytic production of 2-α-d-glucosyl-glycerol for functional ingredient use: integrated process design and techno-economic assessment. ACS Sustainable Chemistry & Engineering 10, 1246-1255 (2022).\nMinor points:\nComment 1: The manuscript should provide a reason to support the assumption of an L-lactate production capacity of 18,500 tons/year.\nReply 1: Thank you for your valuable comment. The projected production capacity of 18,500 tons annually is derived from typical scales used in the commercial lactic acid industry. Most commercial lactic acid production facilities run at capacities ranging from 15,000 to 30,000 tones every year, a range that represents prevailing industry practices, with the exception of a few manufacturers like NatureWorks LLC and Corbion Purac10. Besides, this capacity is frequently employed in techno-economic evaluations of third-generation lactic acid biomanufacturing processes, such as those that utilize carbon dioxide or methane as feedstocks11, 12. Overall, the chosen production capacity conforms to industry standards and guarantees comparability and relevance to existing TEA research. Detailed revision can be found in Line 202-205:\n“It is worth noting that projected production capacity corresponds with standard industrial lactic acid plant scales (15,000-30,000 tons per year)36 and is in accordance with capacities frequently utilized in published TEA studies on third-generation biomanufacturing37, 38.”\nReferences:\n10. Adom, F. K. & Dunn, J. B. Life cycle analysis of corn-stover-derived polymer-grade l-lactic acid and ethyl lactate: Greenhouse gas emissions and fossil energy consumption. Biofuels, Bioproducts and Biorefining 11, 258-268 (2017).\n11. Fei, Q. et al. Biological valorization of natural gas for the production of lactic acid: techno-economic analysis and life cycle assessment. Biochemical Engineering Journal 158, 107500 (2020).\n12. Park, J. et al. Merging biomass and CO utilization; process design and assessment 2 on simultaneous production of lactic acid and formic acid from glycerol and CO . 2 Chemical Engineering Journal 463, 142410 (2023).\nComment 2: It is recommended to improve the visibility of the text and numbers in Figure 6 to enhance overall clarity.\nReply 2: Thank you for your valuable suggestion. In response to your feedback, we have adjusted the font size and formatting of the text and numbers in Fig. 6. The revised version ensures that all elements are easily readable, reducing the potential for misinterpretation.\nComment 3: There is an inconsistency in the use of the terms ‘lactate’ and ‘lactic acid’ between the main text and the supporting information file.\nReply 3: Thank you for pointing out this detail. We have checked the main text and the supporting information file, and changed “lactic acid” to “lactate” in microbial fermentation for consistency. Some specific terms such as poly-lactic acid (PLA) and lactic acid bacteria (LAB) are not changed.\nComment 4: In table S4, there is a typo. ‘Total operating cost’ should be corrected to ‘Total capital cost’\nReply 4: Thank you so much for your advice. As suggested, we have promptly corrected the typo in Supplementary Table S4, changing \"Total operating cost\" to \"Total capital cost\". The revised table has been thoroughly double-checked to ensure its accuracy.\nComment 5: In table S5, which presents the operating expenditures, the use of the term ‘investment’ should be appropriately revised.\nReply 5: Thank you for your comment. Considering that the values in the Supplementary Table S5 represent annual operational expenses, we have carefully amended the term to \"annual costs\" to accurately reflect the nature of these expenditures.\nResponses to Reviewer 6\nGeneral comment: The present manuscript combined three diverse areas in a single paper, lacking thorough justice to individual topics, especially TEA. The TEA used a completely wrong approach for process design, which makes the results unreliable and unacceptable, I recommend removing TEA work from the manuscript to avoid passing the wrong information to the readers. Below are my few comments.\nReply: Thank you for your critical feedback. The TEA and LCA evaluate the entire \"cradle-to-grave\" process of a product or an organization of a certain scale from the perspectives of environmental impact and economic viability. They observe issues from a broad scope, including regional, national, and global levels, and from the perspective of sustainable development, which provide detailed process analysis, argumentation, and assessment for optimization. As a matter of fact, our TEA framework follows to the approach established by the US National Renewable Energy Laboratory (NREL), utilizing publicly accessible models, which are widely used in many TEA-related research1, 2, 13, 14. We sincerely appreciate your comments and hope to address them properly to ensure reliable results for readers.\nReferences:\n1. Zhang, C. et al. An upcycling bioprocess for sustainable aviation fuel production from food waste-derived greenhouse gases: Life cycle assessment and techno-economic analysis. Chemical Engineering Journal 486, 150242, 150242 (2024).\n2. Yan, X. et al. Paradigm of engineering recalcitrant non-model microorganism with dominant metabolic pathway as a biorefinery chassis. Nature Communications 15, 10441, doi:10.1038/s41467-024-54897-5 (2024).\n13. Carpenter, D. et al. Design of an Integrated Solids Handling System to Maximize Syngas Process Reliability (Cooperative Research and Development Final Report). (National Renewable Energy Laboratory (NREL), Golden, CO (United States, 2023).\n14. Kobos, P. H. et al. Techno-economic analysis: Best practices and assessment tools. (Sandia National Lab. (SNL-CA), Livermore, CA (United States); Sandia National, 2020).\nComment 1: Supporting Information: The reaction stoichiometry for methanol production (2 CO2 + 4 H2 → 2 CH3OH + O2) seems to be vague. Also, there are no references to support the reaction conditions for methanol production. Also, I could not find the oxygen as a byproduct.\nReply 1: We are very sorry for making this serious mistake. The correct stoichiometry for methanol production15,16: CO + 3 H → CH OH + H O.\n2 2 3 2\nReferences:\n15. Wang J. et al. A highly selective and stable ZnO-ZrO solid solution catalyst for 2 CO hydrogenation to methanol, Science Advance, 2017, 3, e1701290.\n2\n16. Wang J. et al. High-Performance MaZrOx (Ma = Cd, Ga) Solid-solution catalysts for CO hydrogenation to methanol, ACS Catalysis, 2019, 9, 10253-10259.\nReply 2: The projected production capacity of 18,500 tons annually is derived from typical scales used in the commercial lactic acid industry. Most commercial lactic acid production facilities run at capacities ranging from 15,000 to 30,000 tones every year, a range that represents prevailing industry practices, with the exception of a few manufacturers like NatureWorks LLC and Corbion Purac10. Besides, this capacity is frequently employed in techno-economic evaluations of third-generation lactic acid biomanufacturing processes, such as those that utilize carbon dioxide or methane as feedstocks11, 12. Overall, the chosen production capacity conforms to industry standards and guarantees comparability and relevance to existing TEA research. Detailed revision can be found in Line 202-205:\n“It is worth noting that projected production capacity corresponds with standard industrial lactic acid plant scales (15,000-30,000 tons per year)36 and is in accordance with capacities frequently utilized in published TEA studies on third-generation biomanufacturing37, 38.”\nReferences:\n10. Adom, F. K. & Dunn, J. B. Life cycle analysis of corn-stover-derived polymer-grade l-lactic acid and ethyl lactate: Greenhouse gas emissions and fossil energy consumption. Biofuels, Bioproducts and Biorefining 11, 258-268 (2017).\n11. Fei, Q. et al. Biological valorization of natural gas for the production of lactic acid: techno-economic analysis and life cycle assessment. Biochemical Engineering Journal 158, 107500 (2020).\n12. Park, J. et al. Merging biomass and CO utilization; process design and assessment 2 on simultaneous production of lactic acid and formic acid from glycerol and CO . 2 Chemical Engineering Journal 463, 142410 (2023).\nComment 3: TEA: There is no mention of low pH or neutral fermentation.\nReply 3: Thank you for pointing out this detail. The initial pH was controlled at 5.6 and not further adjusted during cultivation in shake flask. In bioreactor, however, the pH was controlled as constant 5.6 with feeding 4M KOH to neutralize the produced L-lactate. This information was added to Methods section in Line 402-403 and 410-414.\nComment 4: L-Lactate is mentioned in the manuscript. However, the reaction stoichiometry shows L-lactic acid.\nReply 4: Thank you for your comment. L-lactate denotes the conjugate base of L-lactic acid. These terms are frequently used interchangeably in biological contexts depending on pH17. In this study, the fermentation pH was maintained at 5.6. Given that the pKa of lactic acid is 3.86, the produced L-lactic acid was predominantly in its dissociated form, L-lactate. Similar usage occurs in many related studies2,18. Therefore, we used \"L-lactic acid\" in the reaction stoichiometric equations. This distinction has been clarified in the amended “Supplementary Method” section.\nReferences:\n2. Yan, X. et al. Paradigm of engineering recalcitrant non-model microorganism with dominant metabolic pathway as a biorefinery chassis. Nature Communications 15, 10441 (2024).\n17. Pohanka, M. D‐lactic acid as a metabolite: toxicology, diagnosis, and detection. BioMed Research International 2020, 3419034 (2020).\n18. Parra-Ramírez, D., Martinez, A. & Cardona, C. A. Lactic acid production from glucose and xylose using the lactogenic Escherichia coli strain JU15: Experiments and techno-economic results. Bioresource Technology 273, 86-92 (2019).\nComment 5: L-lactate purification (A300): It looks to be completely vague. It may be noted that LA separation is the major cost contributor to the overall process in terms of capital investment and energy consumption. If this kind of downstream separation is considered, the TEA results become completely unreliable. Please refer to recent papers on LA production. Otherwise, I recommend removing TEA completely from the manuscript as it will pass wrong information to the readers.\nReply 5: Thank you for your comments. Our study's design for the L-lactate separation and purification process corresponds to the recent literature — including a dewatering system and a membrane-based separation system (Supplementary Fig. S12) — mirrors approach used in sugar-based and methane-based lactic acid bioprocesses2, 11. Notably, the capital expenditure contribution from the L-lactate separation unit accounts for 12.30% of the total, which falls within the 10–30% range commonly reported in the literature11, 19, 20. Compared to conventional, energy-intensive purification processes (e.g., involving acidification, neutralization, crystallization, evaporation, ion exchange, etc.), membrane separation approach operates under milder conditions with fewer steps, which reduces both energy demand and complexity21, 22. To further clarify this point, we have updated the Supplementary Method and Supplementary Table S2 to provide additional technical details and operational parameters on the L-lactate separation process23-29. Detailed revisions and explanation can be found in Line 202-210:\nMoreover, in numerous high-impact and authoritative international publications30-33, TEA has been extensively utilized to support experimental research by assessing economic viability, hence corroborating findings from an industrial and practical standpoint. Furthermore, it is essential to build the TEA to identify key factors influencing return on investment, thereby directing experiments, and decreasing investment risks34. Consequently, we have preserved the TEA section, including additional clarification and methodological transparency in the updated Supplementary Information to guarantee its rigor and alignment with contemporary research. To further on the significance of TEA, we incorporated the necessary details to the Discussion section on in Line 209-218, 355-357, and 362-363:\n“The CAPEX for equipment and installation of five modules were 2.3%, 59.5%, 12.3%, 7.3%, and 18.6%, respectively (Supplementary Table S4). Comprehensive investment details for equipment and installation corresponding to each individual component are available in Supplementary Fig. S14. Notably, the bioreactors accounted for the predominant share of expenditures associated with equipment and installation in CAPEX. The investment in the purification unit is less extensive due to compared to the traditional separation process, the membrane separation approach operates under milder conditions and involves fewer steps, hence decreasing both energy requirements and complexity39, 40.”\n“The integration of TEA and LCA facilitated a comprehensive assessment of the CO -to-L-lactate 2 biomanufacturing process, revealing essential insights beyond basic experimental performance.”\n“Furthermore, it is imperative to develop the TEA to identify key factors affecting return on investment, thereby guiding experiments, and mitigating investment risks67.”\nReferences:\n2. Yan, X. et al. Paradigm of engineering recalcitrant non-model microorganism with dominant metabolic pathway as a biorefinery chassis. Nature Communications 15, 10441 (2024).\n11. Fei, Q. et al. Biological valorization of natural gas for the production of lactic acid: techno-economic analysis and life cycle assessment. Biochemical Engineering Journal 158, 107500 (2020).\n19. Pal, P., Sikder, J., Roy, S. & Giorno, L. Process intensification in lactic acid production: A review of membrane based processes. Chemical Engineering and Processing: Process Intensification 48, 1549-1559 (2009).\n20. Tejayadi, S. & Cheryan, M. Lactic acid from cheese whey permeate. Productivity and economics of a continuous membrane bioreactor. Applied microbiology and biotechnology 43, 242-248 (1995).\n21. Kumar, R., Basak, B. & Jeon, B.-H. Sustainable production and purification of succinic acid: A review of membrane-integrated green approach. Journal of Cleaner Production 277, 123954 (2020).\n22. Taleghani, H. G., Ghoreyshi, A. A. & Najafpour, G. D. Thin film composite nanofiltration membrane for lactic acid production in membrane bioreactor. Biochemical Engineering Journal 132, 152-160 (2018).\n23. Henderson, R., Parsons, S. A. & Jefferson, B. The impact of algal properties and pre-oxidation on solid–liquid separation of algae. Water research 42, 1827-1845 (2008).\n24. Grima, E. M., Belarbi, E.-H., Fernández, F. A., Medina, A. R. & Chisti, Y. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnology advances 20, 491-515 (2003).\n25. Chen, G., Zhao, L., Qi, Y. & Cui, Y.-L. Chitosan and its derivatives applied in harvesting microalgae for biodiesel production: An outlook. Journal of Nanomaterials 2014, 217537 (2014).\n26. Sikder, J., Roy, M., Dey, P. & Pal, P. Techno-economic analysis of a membrane-integrated bioreactor system for production of lactic acid from sugarcane juice. Biochemical Engineering Journal 63, 81-87 (2012).\n27. Kumar, A., Thakur, A. & Panesar, P. S. Lactic acid and its separation and purification techniques: A review. Reviews in Environmental Science and Bio/Technology 18, 823-853 (2019).\n28. Davis, R. E. et al. Process Design and economics for the conversion of lignocellulosic biomass to hydrocarbon fuels and coproducts: 2018 biochemical design case update; biochemical deconstruction and conversion of biomass to fuels and products via integrated biorefinery pathways. (National Renewable Energy Lab. (NREL), Golden, CO (United States), 2018).\n28. Humbird, D., Davis, R. & McMillan, J. Aeration costs in stirred-tank and bubble column bioreactors. Biochemical engineering journal 127, 161-166 (2017).\n30. Luo, Z. et al. Carbon–carbon bond cleavage for a lignin refinery. Nature Chemical Engineering 1, 61-72 (2024).\n31. Choi, K. R., Luo, Z. W., Kim, G. B., Xu, H. & Lee, S. Y. A microbial process for the production of benzyl acetate. Nature Chemical Engineering 1, 216-228 (2024).\n32. Crandall, B. S. et al. Transforming CO2 into advanced 3D printed carbon nanocomposites. Nature Communications 15, 10568 (2024).\n33. Chi, H. et al. Electrosynthesis of ethylene glycol from biomass glycerol. Nature Communications 16, 979 (2025).\n34. Zhang, C. et al. Economic and sustainable revolution to facilitate one-carbon biomanufacturing. Nature Communications 16, 1-10 (2025).\nComment 6: AD: Reaction stoichiometry and conditions are not mentioned.\nReply 6: Thank you for your comment. This study modelled the anaerobic digestion (AD) unit according to established methodologies reported in the literature, utilizing the RYield block in Aspen Plus, a prevalent technique employed in various process modelling investigations of AD systems35-37. The yield distributions of the AD products were calculated using Fortran code performed by calculator blocks, with residual biomass and waste converted into biogas (CH and CO ), NH , H O, and digestate, in 4 2 3 2 accordance with existing AD process design studies (Supplementary Table S2)35-37. In addition, the AD process operates under mesophilic conditions at 37°C and atmospheric pressure, which are standard conditions for medium-temperature AD37, 38. Detailed AD unit design and parameters are provided in the revised Supplementary Method.\nReferences:\n37. Leal Morais Luna, N. A., Lopes da Silva, M. G., Cezar Menezes, R. S., Dutra, E. D. & Albuquerque, A. A. Simulation refinement of the biogas production process via anaerobic digestion: Validation of Properties, PHASE equilibrium and database construction. Chemical Engineering Science, 121908 (2025).\n38. Wang, T. et al. Biogas-to-methanol: A new green methanol production process based on anaerobic digestion of biomass. Energy Conversion and Management 321, 119065 (2024).\n39. Kelif Ibro, M., Ramayya Ancha, V. & Beyene Lemma, D. Biogas Production Optimization in the Anaerobic Codigestion Process: A Critical Review on Process Parameters Modeling and Simulation Tools. Journal of Chemistry 2024, 4599371 (2024).\n40. Zhang, Z., Zhang, R., Ma, Y. & Sun, Y. Improved volatile fatty acid production in anaerobic digestion via simultaneous temperature regulation and persulfate activation by biochar: Chemical and biological response mechanisms. Environmental Research 264, 120271, doi:https://doi.org/10.1016/j.envres.2024.120271 (2025).\nComment 7: The complete Aspen Plus flowsheet is missing. The details of the equipment and their design parameters are also missing. As a result, it is difficult to judge the reliability of TEA results.\nReply 7: Thank you very much for your constructive comments. We have added the complete Aspen Plus process flowsheet in the Supplementary Fig. S12 to enhance transparency, including essential units and material streams. Additionally, all equipment sizes are directly derived from mass and energy balance data generated by our Aspen Plus model (Supplementary Tables S14-S20). Costs scale with implementation size using a power law model relative to original reference prices, ensuring consistency with industry-standard scaling practices1. Original equipment prices are sourced from the National Renewable Energy Laboratory (NREL) reports and published research30, 31, 41, 42. A detailed breakdown of costs for each unit is provided in Supplementary Fig. S13. The investment ratio in equipment aligns with that of existing biomanufacturing studies and reports1, 43. The detailed revisions can be found in Line 198-202 and 210-215:\n“the proposed production scenario was indicative of the capacity of a plant to produce 18,500 tons of L-lactate annually through three major conversion steps, as illustrated in Supplementary Fig. S12- S13, which depicts the primary carbon flow within the various subsystems and the detailed process design in Aspen plus, respectively.”\n“The CAPEX for equipment and installation of five modules were 2.3%, 59.5%, 12.3%, 7.3%, and 18.6%, respectively (Supplementary Table S4). Comprehensive investment details for equipment and installation corresponding to each individual component are available in Supplementary Fig. S14. Notably, the bioreactors accounted for the predominant share of expenditures associated with equipment and installation in CAPEX.”\nReferences:\n1. Zhang, C. et al. An upcycling bioprocess for sustainable aviation fuel production from food waste-derived greenhouse gases: Life cycle assessment and techno-economic analysis. Chemical Engineering Journal 486, 150242 (2024).\n30. Davis, R. E. et al. Process Design and economics for the conversion of lignocellulosic biomass to hydrocarbon fuels and coproducts: 2018 biochemical design case update; biochemical deconstruction and conversion of biomass to fuels and products via integrated biorefinery pathways. (National Renewable Energy Lab. (NREL), Golden, CO (United States), 2018).\n31. Humbird, D., Davis, R. & McMillan, J. Aeration costs in stirred-tank and bubble column bioreactors. Biochemical engineering journal 127, 161-166 (2017).\n41. Davis, R., Wiatrowski, M., Kinchin, C. & Humbird, D. Conceptual Basis and Techno-Economic Modeling for Integrated Algal Biorefinery Conversion of Microalgae to Fuels and Products (2019 NREL TEA Update: Highlighting Paths to Future Cost Goals via a New Pathway for Combined Algal Processing). (National Renewable Energy Lab. (NREL), Golden, CO (United States), 2020).\n42. Davis, R., McNamara, I. & Bartling, A. Techno-Economic Case Study: Low-Temperature Conversion Performance Based on Isolated Anatomical Fractions of Corn Stover. (National Renewable Energy Laboratory (NREL), Golden, CO (United States), 2023).\n43. Shaji, A., Shastri, Y., Kumar, V., Ranade, V. V. & Hindle, N. Economic and environmental assessment of succinic acid production from sugarcane bagasse. ACS Sustainable Chemistry & Engineering 9, 12738-12746 (2021).\nComment 8: The energy cost is completely missing from TEA, which is one of the main cost contributors in this process.\nReply 8: Thank you for your comment. The energy costs are incorporated into the operating expenditures (OPEX). We calculated power and energy requirements based on the Aspen Plus simulation, and the associated data have been included in Supplementary Tables S19-S20. Following the implementation of the heat integration method, energy consumption is diminished by more than 95%, consistent with prior studies42, 43. The combined heat and power (CHP) unit generates 24,502 kW of electricity, satisfying 46.15% of the plant's operational energy needs. Hence, we considered the cost of power consumption, and the facility's overall annually power expenditures reach 7.02 million dollars (Supplementary Tables S5).\nReferences:\n42. Kim, H., Baek, S. & Won, W. Integrative technical, economic, and environmental sustainability analysis for the development process of biomass-derived 2,5-furandicarboxylic acid. Renewable and Sustainable Energy Reviews 157, 112059 (2022).\n43. Jeong, S., Kim, J., Liu, J. J. & Won, W. A lignocellulosic 2,3-butanediol strategy: Process development and integrative analyses. Journal of Environmental Chemical Engineering 11, 110085 (2023).\nComment 9: “The estimation of CAPEX, OPEX, and MSP was conducted via an in-house model created by the National Renewable Energy Laboratory (NREL).” Is it an open-source software? Where is the link?\nReply 9: Thanks for your comments. The estimation of CAPEX, OPEX, and MSP in our study is based on the original calculation spreadsheets and models developed by the NREL, which are widely used in TEA-related research1, 2, 11, 13, 14. These tools are open-source and publicly accessible and the resources14 can be downloaded from the NREL website at: https://www.nrel.gov/analysis/techno-economic.html. The relevant description is detailed in the Methods section of the manuscript in Line 490-491 as follows:\n“The estimation of CAPEX, OPEX, and MSP was conducted via an in-house model created by the National Renewable Energy Laboratory (NREL).”\nReferences:\n1. Zhang, C. et al. An upcycling bioprocess for sustainable aviation fuel production from food waste-derived greenhouse gases: Life cycle assessment and techno-economic analysis. Chemical Engineering Journal 486, 150242 (2024).\n2. Yan, X. et al. Paradigm of engineering recalcitrant non-model microorganism with dominant metabolic pathway as a biorefinery chassis. Nature Communications 15, 10441 (2024).\n11. Fei, Q. et al. Biological valorization of natural gas for the production of lactic acid: techno-economic analysis and life cycle assessment. Biochemical Engineering Journal 158, 107500 (2020).\n13. Carpenter, D. et al. Design of an Integrated Solids Handling System to Maximize Syngas Process Reliability (Cooperative Research and Development Final Report). (National Renewable Energy Laboratory (NREL), Golden, CO (United States), 2023).\n14. Kobos, P. H. et al. Techno-economic analysis: Best practices and assessment tools. (Sandia National Lab. (SNL-CA), Livermore, CA (United States), 2020).\nResponses to Reviewer 7\nGeneral comment: The article entitled “ Methanol biotransformation for the production of the biodegradable plastic monomer L-lactate in yeast” discusses a promising approach to producing L-lactate, a biodegradable plastic monomer via methanol biotransformation in yeast Ogataea polymorpha. The topic is highly relevant and of significant interest, particularly in the context of use of C1 feedstock for sustainable biomanufacturing of plastics. However, the Manuscript needs lots of clarity as there are several loopholes. Below are my comments and suggestions to enhance the quality of the Manuscript.\nReply: We appreciate your positive comments on our work. Here, we provide a point-by-point reply to address your concerns.\nComment 1: Throughout the Manuscript, the argument of author is that methanol is an ideal feedstock for biomanufacturing lactate (LA) as it can be produced from CO in 2 massive quantities and this approach will help in mitigating the carbon emissions. In that case why not to use CO directly via gas fermentation instead of methanol. I would 2 suggest adding some text for comparing LA production using CO and methanol with 2 pros and cons of each and demonstrate which one could be more suitable to take forward.\nReply 1: Thank you for your valuable suggestion. Although CO can be used directly 2 to produce lactate, the titer and productivity are much lower than that from methanol (Table 1). The main reason is the low gas-to-liquid transfer rate in gas fermentation. In comparison, the high liquid-to-liquid transfer rate in methanol fermentation greatly facilitates microbial cell growth and chemicals production. We have added this description in the Discussion (Line 290-293).\nComment 2: Usually most of the microbial cultures have lactate dehydrogenase enzymes. Does genome of Ogataea polymorpha has no copy of this enzyme? Add few lines on Biochemistry and Microbiology of yeast.\nReply 2: Thank you for your valuable suggestion. While O. polymorpha, like other yeasts such as Saccharomyces cerevisiae and Pichia pastoris, possesses a LDH gene in its genome, these yeasts naturally produce no lactate. Lactate production only occurs when a bacterial LDH gene is introduced and expressed. We speculate that the native yeast LDH may encode an enzyme catalyzing the reverse reaction (lactate to pyruvate), consistent with their known ability to degrade lactate. To enable lactate production in O. polymorpha from methanol, we screened five LDH genes from different LAB. Among these, the Ldh from P. acidilactici demonstrated the highest performance for L-lactate production. We have added this description to the Discussion in Line 309-315.\nComment 3: The role of LPL1 (encoding a putative lipase) and IZH3 (encoding a membrane protein) in context of LA production is not clear?\nReply 3: Thank you for pointing out this detail. In our previous study, we found that mutation of IZH3 and LPL1 was responsible for growth recovery of fatty acid producing strain which cannot grow in methanol. LPL1 possesses the activity of phospholipase/lysophospholipase for degrading various phospholipid substrates and IZH3 is a membrane protein related to zinc metabolism. izh3Δ mutation was found to alleviate the DNA damage caused by antimicrobial peptide (Molecular Microbiology, 2007, 65: 494-507), and it is speculated that the izh3Δ mutation may also attenuate the damaging effects of formaldehyde during L-LA production. In contrast, the disruption of LPL1 blocked phospholipid degradation, which may direct more carbon flux from pyruvate to acetyl-CoA to synthesize lipid, therefore the flux from pyruvate to L-lactate was reduced. We have added this description to the Discussion in Line 320-329.\nComment 4: Why and how ALE was done, what was the selection pressure. It was not clear how ALE cause reduction in LA production despite substantial improvement in biomass.\nReply 4: The detailed process of ALE was described in Methods in Line 386-392. To be specific, the selection pressure is cell growth under methanol stress (10 g/L methanol). We intended to increase methanol tolerance of the L-lactate producing strain, so as to increase cell growth, as well as L-lactate titer. While the methanol tolerance of O. polymorpha was easy to increase, which was inflected by the obviously improved cell growth, the L-lactate titer dramatically decreased. We supposed that the better cell growth redirected more carbon flux from pyruvate to TCA cycle, therefore the flux to L-lactate was severely reduced. In other words, the cell growth competes pyruvate with L-lactate synthesis in methanol fermentation.\nComment 5: If LA production is be taken place in mitochondria where plenty of NADH is available from TCA cycle, then why do we need NADPH as cofactor?\nReply 5: The engineered strain HPLA22 has dual L-lactate producing pathways in cytoplasm (using NADPH as cofactor) and mitochondria (using NADH as cofactor). Therefore, both NADPH in cytoplasm and NADH in mitochondria were needed to produce L-lactate production from methanol.\nComment 6: First of methanol is a reduced carbon source and should generate more reducing equivalents than glucose. Even if it is NADPH, transhydrogenase can easily convert them into NADH.\nReply 6: Indeed, methanol has higher reduction degree than glucose. However, in methylotrophic yeast, alcohol oxidase (Aox) oxidizes methanol to formaldehyde, and the H reacts with O to form H O . Then H O is catalyzed by catalase (Cat) to H O 2 2 2 2 2 2 and O . Therefore, the reduction equivalent (H) in methanol metabolism is wasted. 2 Since the cofactors NADH and NADPH drive numerous cellular reactions, introducing transhydrogenase to convert NADPH to NADH could disrupt native cofactor distribution and trigger destructive effects on overall metabolism. To isolate the impact of Ldh and avoid unknown interactions from altered redox balances, we focused exclusively on expressing Ldh without co-expressing transhydrogenase.\nComment 7: It is difficult to buy this argument that due to methanol metabolism, NADH is not available for LA production.\nReply 7: Sorry for the confusing description. We wish to clarify the cofactor distribution in our system. Under methanol metabolism, the NADPH/NADP⁺ pool is substantially more abundant than the NADH/NAD⁺ pool. This NADPH abundance makes it a more suitable cofactor for L-lactate production via Ldh. Indeed, while strain HPLA06 (utilizing NADH) produced 1.2 g/L L-lactate, strain HPLA13 (engineered to utilize NADPH) achieved a significantly higher titer of ~1.6 g/L L-lactate. This confirms NADPH's superior driving force for lactate synthesis in this study.\nComment 8: Usually LA production takes place in cytoplasm as pyruvate comes from Glycolysis which happen in cytoplasm. In current study, it is not clear why LA production was shifted to mitochondria.\nReply 8: In this study, we introduced Ldh enzyme which is fused with mitochondrial tarting signal (MTS) to O. polymorpha. The MTS peptide can target Ldh from cytoplasm to mitochondria, thus enabling L-lactate production in mitochondria. As the final engineered strain HPLA22 has both Ldh and MTS-Ldh, the L-lactate production from methanol take place in both cytoplasm and mitochondria.\nComment 9: The Manuscript does not about oxygen levels during cultivation. For example, I could not find any information about aeration conditions during fed-batch culture.\nReply 9: Thank you for pointing out this detail. In the fed-batch fermentation in bioreactor, the pH, temperature, and DO were set as 5.6, 37°C, and 30%, respectively. Initial agitation was set to 400 rpm, and increased to maximal 800 rpm, depending on the DO level. Aeration was initially provided at 18 sL/h and increased to maximally 48 sL/h depending on DO level. We have added these conditions to Methods in Line 410-414.\nComment 10: The batch and fed-batch fermentation were performed only on methanol or some glucose was added during the cultivation?\nReply 10: All fermentations for L-lactate production in this study used methanol as the sole carbon source without any auxiliary carbon sources.\nComment 11: What is the theoretical yield of LA on methanol. Were there any byproducts formed? What was the pH profile in flask and was pH controlled in bioreactor?\nReply 11: We calculated the theoretical yield of L-lactate on methanol is 0.804 (g/g). A key advantage of O. polymorpha is its clean metabolic profile: it produces minimal byproducts, with only trace amounts of intermediates (e.g., TCA cycle organic acids or formate) detectable. The pH was controlled initially at 5.6 and not further adjusted during cultivation in shake flask. In bioreactor, the pH was controlled at constant 5.6 with feeding 4M KOH to neutralize the produced L-lactate.\nComment 12: The details of software used for LCA and TEA is not provided. Despite a LA titer of 25 g/L, MSP is coming out to be $2.25/kg which is better than many sugar based bioprocesses. I am not sure how they are ending up with such a low number.\nReply 12: The software used for LCA and TEA was described in the Methods section of the original manuscript (“TEA and LCA analysis”) in Line 478-480, 490-491 and 530-531. All data used in both analyses were derived from a rigorously developed process simulation in Aspen Plus v14. LCA was performed using openLCA v2.2 (www.openlca.org), while TEA was conducted using an in-house model based on the open-source National Renewable Energy Laboratory TEA framework (https://www.nrel.gov/analysis/techno-economic.html)14. Regarding the minimum selling price (MSP) of L-lactate, we have included a comparison with sugar-based lactic acid production in the Supplementary Table S9. The MSP for sugar-based lactic acid ranges from $0.35-1.57 /kg2, 44-46, which is generally lower than the value reported in our study. A detailed analysis of the comparison between MSP and sugar-based lactic acid can be found in Line 357-361.\nReferences:\n2. Yan, X. et al. Paradigm of engineering recalcitrant non-model microorganism with dominant metabolic pathway as a biorefinery chassis. Nature Communications 15, 10441, doi:10.1038/s41467-024-54897-5 (2024).\n14. Kobos, P. H. et al. Techno-economic analysis: Best practices and assessment tools. (Sandia National Lab. (SNL-CA), Livermore, CA (United States), 2020).\n44. Liu, G., Sun, J., Zhang, J., Tu, Y. & Bao, J. High titer L-lactic acid production from corn stover with minimum wastewater generation and techno-economic evaluation based on Aspen plus modeling. Bioresource Technology 198, 803-810 (2015).\n45. Kwan, T. H., Hu, Y. & Lin, C. S. K. Techno-economic analysis of a food waste valorisation process for lactic acid, lactide and poly (lactic acid) production. Journal of cleaner production 181, 72-87 (2018).\n46. Li, Y. et al. Sustainable lactic acid production from lignocellulosic biomass. ACS Sustainable Chemistry & Engineering 9, 1341-1351 (2021).\n“This study entailed the design of an L-lactate production process using Aspen Plus v14 software, which was utilized to conduct calculations for mass and energy balance.”\n“The estimation of CAPEX, OPEX, and MSP was conducted via an in-house model created by the National Renewable Energy Laboratory (NREL).”\n“All calculations and estimations are predicated on openLCA software version 2.2.”\n“Although the competitive potential is inadequate to that of established sugar-based and lignocellulosic processes (Supplementary Table S9), this study emphasize the importance of third-generation biomanufacturing — particularly regarding reduced carbon source costs, optimized process utility, and carbon credit considerations — which collectively improve economic viability, resulting in an MSP lower than the market price of L-lactate.”\nResponses to the reviewers’ comments (NCOMMS-25-14369)\nDear reviewers,\nWe would like to express our thanks to the constructive suggestions regarding to our manuscript “Methanol biotransformation for the production of the biodegradable plastic monomer L-lactate in yeast”. These comments are helpful for us to revise and further improve our paper. The main corrections and responses to the reviewers’ comments are as follows:\nReviewer #5:\nGeneral comment\nBiodegradable polymers like polylactic acid (PLA) are increasingly recognized as eco‑friendly substitutes for petroleum-based plastics, and novel single‑carbon (C1) bioprocesses present an efficient pathway for lactate synthesis. The yeast Ogataea polymorpha was genetically modified to achieve high yields of L‑lactate solely from methanol. The authors introduce an integrated approach that combines “liquid sunshine” photochemical conversion with engineered O. polymorpha fermentation to enhance L-lactate titers, and assess the system’s techno-economic viability alongside its greenhouse-gas reduction potential. I think this paper can be published in Nature Communications after the second revision for one major point.\nResponse: We sincerely appreciate the reviewer for the constructive and encouraging comments on our manuscript. We have carefully addressed the major concern raised and revised the manuscript accordingly. We hope that these improvements will address your concerns.\nMajor points\nComment 1. Thank you for the revision and efforts to address the comment. The authors explained that the levelized cost of electricity for utility-scale photovoltaic solar power in 2024 is approximately $0.031-0.075/kWh with references. However, some references, such as the Fraunhofer report (2024), present the levelized cost of electricity for photovoltaic solar power as a range of $0.04-0.24/kWh. It is recommended to consider the various ranges of the levelized cost of photovoltaic solar power, rather than focusing solely on the minimum cost. Considering that photovoltaic solar power is a form of renewable energy, its price varies greatly depending on the region, making it infeasible to reflect the minimum price in a specific region. The authors should consider the price of photovoltaic solar power in various regions and perform the techno-economic analysis for two cases: when the solar power cost is minimum and when it is maximum. The Fraunhofer report (2024): Christoph Kost et al., Levelized Cost of Electricity Renewable Energy Technologies, Fraunhofer, 2024.\nReply 1: Thank you for your detailed comment. In the revised manuscript, we incorporated sensitivity analysis scenarios by using utility-scale photovoltaic power prices from different regions, drawing on multiple references including Fraunhofer (2025)1-6. A corresponding sensitivity curve is provided in Supplementary Fig. S15. We also explicitly discussed the regional variations in photovoltaic electricity prices in the manuscript (page 10, lines 248-255):\n“Considering the regional variation in solar power costs, the MSP fluctuates significantly with the geographical location of the plant43 (Supplementary Fig. S15). For example, countries with abundant solar resources such as Pakistan show solar power prices close to the global minimum (<0.025 $/kWh), resulting in comparatively lower MSP, whereas regions with higher solar power prices such as Canada (>0.10 $/kWh) may increase the MSP by over 13%. These results emphasize the critical role of site selection in improving the economic competitiveness of CO -to-L-lactate 2 biomanufacturing.”\nSupplementary Fig. S15 Regional impact of utility-scale photovoltaic solar levelized cost of electricity (LCOE) on the minimum selling price of L-lactate in 2024. Data sources: Pakistan1, India2, global average2, United States3, Southern Germany4, China5, Northern Germany4, Canada6.\nReviewer #1 (Remarks to the Author):\nGeneral comment\nThe authors have addressed all questions and comments satisfactorily. I am fine with the current version and support its publication. There are just a few minor writing issues that should be corrected.\nMinor corrections\nLine 111: in situ should not be italicized.\nLine 160: Please be mindful of the tense in this sentence (\"It is worth mentioning...\").\nReply: Thank you for your valuable suggestions. We have corrected the mistakes in Line 111 and 160."}]

Decision letter