Engineering Yeasts to Grow Solely on Methanol or Formic acid coupled with CO2 fixation

doi:10.21203/rs.3.rs-2694097/v1

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Engineering Yeasts to Grow Solely on Methanol or Formic acid coupled with CO₂ fixation

https://doi.org/10.21203/rs.3.rs-2694097/v1

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Synthetic microorganisms capable of using one-carbon compounds, such as methanol, formic acid or carbon dioxide, are of increasing interest. In this study, we converted the yeasts of Pichia pastoris and Saccharomyces cerevisiae to both synthetic methylotroph and formatotroph, allowing them to grow on methanol and formic acid alone coupled with CO₂ fixation through a synthetic C1-compound assimilation pathway (MFORG pathway). This pathway consists of a methanol-formic acid oxidation module and the reductive glycine pathway. We first assembled the MFORG pathway in P. pastoris using only native enzymes, followed by overexpression of genes in the reductive glycine pathway, blocking the native methanol assimilation pathway, and compartmentalizing the methanol oxidation module. These modifications successfully redesigned the native methylotrophic yeast P. pastoris to grow on both methanol and formic acid, where higher growth rate and yield on methanol was obtained compared to the wild-type strain. We then introduced the MFORG pathway from P. pastoris into the model yeast S. cerevisiae, establishing full synthetic methylotrophy and formatotrophy in this organism. The resulting strain was able to successfully grow on methanol or formic acid alone with consumption rates of 24 mg/L*h and 15.2 mg/L*h, respectively. The CO₂ fixation ability of synthetic P. pastoris and S. cerevisiae through the MFORG pathway was confirmed by ¹³C-tracer analysis. Finally, production of 5-aminolevulinic acid and lactic acid with methanol as the sole carbon source was demonstrated using synthetic P. pastoris and S. cerevisiae, indicating the potential of yeasts as promising hosts for biochemical production from various one-carbon compounds.

Biological sciences/Biotechnology/Metabolic engineering

Biological sciences/Biochemistry/Metabolomics

Synthetic C1-compound assimilation pathway

Pichia pastoris

Saccharomyces cerevisiae

Methanol

Formic acid

With increasing concerns surrounding climate change, biological carbon capture and utilization technologies have recently attracted widespread attention^{1, 2, 3, 4}. In addition to direct CO₂ fixation, the biological assimilation of the liquid one-carbon (C1) compounds, such as methanol or formic acid can be a viable alternative for converting CO₂ to value-added products^{5, 6, 7, 8, 9}. These C1-compounds can be easily and efficiently synthesized from CO₂ by various chemical processes which have been explored in several studies^{10, 11, 12}. These C1-compounds have been considered ideal fermentative feedstocks, which may relieve the industrial dependence on sugar-containing raw materials and also be beneficial for global carbon neutrality^{13, 14, 15}. Besides serving as a carbon source, liquid C1-compounds are also promising energy sources due to their higher reduction capacity per carbon than most sugars, making them potential electron donors to drive CO₂ fixation^{16, 17}. Recently, the development of a synthetic microorganism capable of co-utilization of CO₂ and liquid C1-compounds has received increased attention.

Two alternatives have emerged for co-utilization of CO₂ and liquid C1-compounds. One option is to develop a synthetic autotroph with liquid C1-compounds as the energy source instead of carbon source¹⁸. For example, using formic acid as the energy source, an autotrophic E. coli expressing the Calvin-BensonBassham (CBB) cycle were constructed for CO₂ fixation¹⁹. In addition, P. pastoris was converted into an autotroph growing on CO₂ by resembling the CBB cycle, wherein methanol was used as a supply of reducing power¹⁶. Generally, the oxidation rate of formate or methanol was higher than the rate at which CO₂ was assimilated, and growing these engineered autotrophic strains on formate or methanol might lead to an overall net CO₂ production. Accordingly, the development of mixotrophic methylotrophs or formatotrophs in which liquid C1-compounds are both used as the carbon source and energy source for CO₂ fixation has been pursued with the potential for increased carbon efficiency^{6, 19, 20, 21, 22}. The reductive glycine pathway, a linear route for directly assimilating formate and CO₂ into the central metabolism, has been commonly engineered for C1 compound co-utilization²³. As a result, several mixotrophic E. coli strains able to grow on CO₂ and formic acid alone have been produced^{20, 21, 24}. One mixotrophic E. coli strain capable of growth on CO₂ and methanol alone was also reported, however, the growth rate was extremely low due to limited methanol oxidation rate²¹. Recent efforts are often limited to the E. coli model. Expanding the range of microorganisms able to co-utilize CO₂ and methanol or formic acid for bioproduction is therefore highly desirable.

Yeasts are ideal hosts for biotechnological production processes as they possess advantages in protein expression as well as high tolerance to harsh fermentation conditions^25,26. In contrast to bacterial methylotrophs, yeasts capable of growing on methanol or formic acid, as well as CO₂ fixation have received only limited attention in biotechnological research. P. pastoris is a native methylotroph. In recent years, efforts in engineering P. pastoris to synthesize valuable metabolites from C1-compounds are limited to the use of methanol as the sole carbon source^{27, 28, 29}. C1-compund-utilisation research in S. cerevisiae is still in its infancy. In particular, synthetic methanol or formic auxotrophy has not been demonstrated in S. cerevisiae although the entire methanol pathway from P. pastoris or methylotrophic bacteria has been successfully introduced¹⁸.

In this study, we reported the establishment of both synthetic methylotrophy and formatotrophy in P. pastoris and S. cerevisiae to enable them grow solely on methanol and formic acid coupled with CO₂ fixation via a synthetic C1 assimilation pathway in and the methanol and formic acid oxidation module with the reductive glycine pathway (MFORG pathway). First, in P. pastoris, the native methanol assimilation pathway of the xylulose monophosphate (XuMP) cycle was blocked and replaced with the MFORG pathway. It was then engineered to co-assimilate methanol and CO₂ by optimizing the expression of different modules coupled with a compartmentalization strategy. Subsequently, the entire reconstituted MFORG pathway was transplanted into S. cerevisiae, which converted S. cerevisiae into a full synthetic methylotroph able to grow on methanol for CO₂ fixation. As formic acid was an intermediate in the MFORG pathway, the growth of engineered P. pastoris and S. cerevisiae on formic acid as the sole carbon source was also achieved and characterized. The production of 5-aminolevulinic acid and lactic acid using these engineered P. pastoris and S. cerevisiae growing on methanol alone was also demonstrated.

The synthetic methanol and formic acid oxidation-reductive glycine pathway

In this study, we aimed to design and reconstruct a synthetic pathway capable of supporting yeast growing solely on methanol and formic acid, as well as the co-assimilation of CO₂. The feasibility of approach in converting a non-native methylotroph to a synthetic methylotroph was also evaluated. P. pastoris is a model methylotrophic yeast capable of growth on methanol alone. The natural methanol metabolism in P. pastoris involves carbon dioxide loss via the formaldehyde dissimilatory pathway through glutathione-dependent formaldehyde dehydrogenase (FLD), S-formyl glutathione hydrolase (FGH), and formate dehydrogenase (FDH), the main source of cellular redox power for support of cell growth. The inactivation of formaldehyde dissimilation for carbon conservation through deletion of FLD or FDH would significantly reduce the biomass yield of P. pastoris on methanol³⁰. The transfer of the entire methanol utilization pathway in P. pastoris into other model yeasts including S. cerevisiae and Yarrowia lipolytica has been demonstrated to be a difficult and inconvenient strategy in developing full synthetic methylotrophic yeast.

Here, we constructed a synthetic linear route called the methanol and formic acid oxidation-reductive glycine pathway (MFORG) for assimilating methanol or formic acid with CO₂ fixation into biomass. This pathway is modified from the combination of methanol and formic acid oxidation modules with the reductive glycine pathway (Fig. 1). The pathway is divided into four modules as illustrated in Fig. 1. The first module is methanol oxidation, consisting of alcohol oxidase (AOX), FLD, and FGH, responsible for converting methanol to formic acid and providing the cell with reducing power and energy. The second module is formic acid oxidation consisting of FDH, converting formic acid to CO₂ in order to generate another equivalency of reducing power to support cell growth. The third module is C1 compound co-assimilation, consisting of C-1-tetrahydrofolate synthase (MIS1), aminomethyl transferase (GCV1), and glycine cleavage system H protein (GCV3) which collectively condense formic acid with CO₂ and ammonia to produce glycine. The last module is pyruvate synthesis, consisting of serine hydroxymethyl transferase gene (SHM1), and L-serine dehydratase (CHA1). The reductive glycine pathway (rGly), includes the two downstream modules, and is a critical part of the designed MFORG pathway²³. It has recently been identified as the most efficient route of formate assimilation under aerobic conditions. The core of the rGly pathway is the conversion of formate, CO₂, and NH₃ to glycine²³. When further combined with methanol oxidation to formic acid, the pathway may also be altered to co-assimilate methanol and CO₂, providing a flexible platform for bioconversion of various C1-compounds. Based on this pathway, engineered E. coli strains growing formic acid, methanol, and CO₂ alone have been developed^{21, 24}. The core module of the rGly pathway has also been engineered in S. cerevisiae to convert formate to glycine³¹. However, growth of the yeast on liquid C1-compounds alone remains an open challenge.

Reconstruction and optimization of the MFORG pathway in Pichia pastoris

To facilitate establishment of the MFORG pathway to obtain flexible C1-compound-assimilating yeast, we began with the model methylotrophic yeast of P. pastoris. We started with a P. pastoris strain of PMORG01 deficient in the native methanol assimilatory pathway through deletion of the genes encoding for DAS1 and DAS2. This initial strain showed no growth over the entire cultivation phase on methanol and was therefore used as a negative control (Fig. 2A), which indicated that the activities of endogenous enzymes in the MFORG pathway were too low to support the required flux. As one of most important parts of the MFORG pathway, the genes encoding MIS1, GCV1, GCV2, and GCV3 in the C1-compound co-assimilation module were first over-expressed to obtain the strain PMORG02. As illustrated in Fig. 2A, although the methanol consumption rate and final biomass titer of PMORG02 were all slightly lower than those of the wild-type GS115, the ability of strain PMORG02 to grow on methanol alone was successfully achieved, indicating the feasibility of MFORG pathway for methanol assimilation in P. pastoris. Over the course of the 144-hour fermentation, PMORG02 grew to an OD₆₀₀ of 0.5.

In the MFORG pathway, both the FLD in the methanol oxidation module and FDH in the formic acid oxidation module generated reducing power and energy to support cell growth when using methanol as the carbon source. In the second reaction catalyzed by FDH, formic acid is converted to CO₂ and induces CO₂ emission. The effect of inactivating the formic acid oxidation module on cell growth in engineered P. pastoris PMORG02 was therefore evaluated. The results are shown in Fig. 2C. The overexpression of genes in the C1-compound co-assimilation module in a PMORG04 strain also supported cell growth on methanol alone. However, deletion of FDH significantly reduced the growth rate and final biomass by 20% and 5% respectively. In the C1-compound co-assimilation and pyruvate synthesis module, 2 mol of ATP and 3 mol NAD(P)H were required to convert formic acid and CO₂ into pyruvate (Supplementary Table 1). Based on a stoichiometric analysis, deletion of FDH induced insufficient reducing power supply required for formic acid assimilation and decreased cell growth. As the results we observed, formic acid accumulated due to the deletion of FDH, especially in the MFORG-expressing P. pastoris PMORG04 strain (Fig. 2B).

The lower growth of MFORG-expressing P. pastoris compared to wild-type GS115, was analyzed and the effects of the methanol oxidation module and pyruvate synthesis module on methanol assimilation were investigated to further improve the pathway efficiency. In the C1-compound co-assimilation module, all reactions from formate to glycine are fully reversible. Therefore, from a thermodynamic perspective, high concentrations of formate are beneficial for pathway efficiency²⁰. To improve formic acid supply, the enhancement of the methanol oxidation module was implicated. In P. pastoris, the process for methanol oxidation to formaldehyde is localized to the peroxisome³². This subcellular compartmentalization has some advantages, such as enriching substrate for enzyme access and relieving the toxicity of the intermediate formaldehyde. As high concentrations of formic acid are also toxic to yeast, we used a compartmentalization strategy to relocate the entire methanol oxidation module into the peroxisome while overexpressing FLD and FGH to improve formic acid generation (Fig. 3A). Subcellular localization of FLD and FGH into the peroxisome using the PTS1 signal peptide was experimentally confirmed by fusing fluorescent protein (GFP or RFP) to these two proteins in the PMFORG2 strain (Fig. 3B). Compared to the PMFORG2 strain, overexpression of FLD and FGH in the cytoplasm (PMFORG5) was able to improve biomass production by 11% (Fig. 3C). Furthermore, targeting the methanol oxidation module to the peroxisome (PMFORG6) increased biomass production by 15% compared to the PMFORG2 strain. The methanol consumption rate was also slightly increased due to the engineering of methanol oxidation module (Fig. 3C). Beginning from the FDH-deficient strain PMFORG4, peroxisomal expression of the methanol oxidation module also produced a significant increase in cell growth on methanol by 15.9%, and increased formic acid consumption (Fig. 3C). The biomass yield of PMFORG6 was slightly higher than the wild-type strain GS115 when cultured in minimal medium with methanol as the sole carbon source (Table 1).

Table 1

Methanol consumption rate, formic acid accumulation, biomass production, doubling time, and CO₂ fixation rate of GS115, PMORG02, PMORG04, PMORG06, PMORG08, PMORG09, and PMORG11 strains
Strain	Methanol consumption rate (mg/h*L)	Formic acid accumulation (g/L)	Biomass yield (gDCW/moL methanol)	Doubling time(h)	CO₂ fixation rate* (g/h*L)
GS115	41.67	0.00	0.77	87	-
PMORG02	48.50	0.00	0.72	73	0.033
PMORG04	40.60	1.01	0.71	92	0.016
PMORG06	49.90	0.00	0.80	61	0.034
PMORG08	42.60	0.60	0.78	93	0.018
PMORG09	52.00	0.00	0.89	59	0.314
PMORG11	44.70	0.48	0.85	64	0.018
*The specific CO₂ consumption rate is not a measured value. It is calculated from the methanol or formic acid consumption rate using the stoichiometry of the C1 assimilation pathway; the C1 assimilation pathway assimilates two molecules of methanol or formic acid and one CO₂ molecule.

We further targeted the downstream pyruvate synthesis module by overexpressing the SHM1 and CHA1 gene. To better investigate the effect of the pyruvate synthesis module on methanol utilization, we constructed strains of PMORG09 and PMORG10 beginning from PMORG06, which overexpressed SHM1 or SHM1-CHA1 respectively. As shown in Fig. 3C, PMORG09 has enhanced growth in minimal salt medium with methanol as the sole carbon source, which increased biomass production by 13.2% compared to PMORG06 without the enhanced downstream pathway. However, PMORG10 co-expressing SHM1 and CHA1 genes did not grow well, which may be related to the reversible reaction catalyzed by CHA1. Overexpression of SHM1 moderately increased the methanol consumption rate (Fig. 3C, Table 1). Beginning with an FDH-deficient strain PMFORG4, overexpression of genes in the pyruvate synthesis module had a similar effect on cell growth. In summary, the engineered P. pastoris strain was able to grow on methanol solely based on the synthetic MFORG pathway. After optimizing gene expression in the MFORG pathway, the final strain PMORG09 exhibited significant advantages in methylotrophy capacity, where growth rate, biomass yield, and methanol consumption rate were 1.43-fold, 1.16-fold, and 1.25-fold higher compared to wild-type GS115 (Fig. 3D, Table 1).

Incorporation of C1 compound verified by ¹³ C-tracer analysis in engineered P. pastoris

In the previous study, the rGly pathway, the downstream segment in our synthetic MFORG pathway, was established in E. coli to grow on methanol or formic acid alone. However, this growth was dependent on a high CO₂ concentration (10% CO₂) due to the low affinity towards inorganic carbon of enzymes in the rGly pathway²³. Surprisingly, our recombinant P. pastoris strain expressing the MFORG pathway was able to grow successfully on methanol without the extra addition of high CO₂ concentration. Specifically, a certain concentration of CO₂ negatively affected cell growth on methanol (Supplementary Fig. 1). In the methanol oxidation module, methanol is catalyzed by alcohol oxidase (AOX) to produce formaldehyde, and this reaction process requires a large amount of oxygen³³. The introduction of a large amount of CO₂ may inevitably reduce oxygen content, thus reducing the efficiency of methanol utilization by the strain.

To verify whether CO₂ could be also fixed through the MFORG pathway, we fed cultures of strains GS115, PMFORG6, and PMFORG9 with ¹²C-methanol and ¹³C-CO₂ in minimum salt medium and determined the labeling pattern of proteinogenic amino acids by gas chromatography-time-of-flight mass spectrometry (GC-TOF-MS). A total of nine amino acids including glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), aspartic acid (Asp), Glutamic Acid (Glu), valine (Val), leucine (Leu), and isoleucine (Iso) were detected and found to be labeled by¹³C-CO₂-derived carbons. As expected, the MFORG-expressing strains of PMORG06 and PMORG09 had a significantly increased labeling in all nine amino acids compared to the wild-type GS115, especially Gly and Ser, the intermediates of MFORG pathway. For example, the average carbon labeling percentage of Gly was increased to 40% in AMFORG6, and 42% in PMFORG9, approximately 2-fold higher than observed in the wild-type GS115. As these amino acids are all derived from the central carbon metabolism, their labeling indicated that the engineered P. pastoris was able to fix CO₂ to enter the central metabolism via the MFORG pathway to synthesize relevant intermediates when growing on methanol solely. We found that the wild-type strain was labeled with a small number of amino acids in the medium supplemented with ¹³C-CO₂, suggesting the presence of an endogenous CO₂-fixing process in yeast.

To further confirm that the MFORG-expressing P. pastoris could co-assimilate methanol and CO₂, we performed carbon-labeling experiments with PMFORG9 providing: (i) ¹³C-CO₂ and unlabeled methanol, (ii) ¹³C-methanol and unlabeled CO₂, or (iii) ¹³C-methanol and ¹³C-CO₂. As the results indicated in Fig. 4B, 4C and 4D amino-acid labeling confirmed the ability to co-assimilate methanol and CO₂ of the synthetic MFORG pathway. A total of nine different amino acids were labeled by ¹³C. When feeding with ¹³C-methanol and ¹³C-CO₂, the labeled level of these detected amino acids was over 85% (Fig. 4D). The values of some amino acids, such as Ser, Thr, and Asp, were more than 96% (Fig. 4D). More importantly, the co-feeding of ¹³C-methanol and ¹³C-CO₂ significantly increased ¹³C-labeled levels of different amino acid compared to those when fed with ¹³C-methanol or ¹³C-CO₂ alone. For example, 82% of Ser was labeled when ¹³C-methanol was supplemented, and 67% of Ser was labeled in the presence of ¹³C-CO₂ alone (Fig. 4B and 4C). However, the labeled Ser could reach 98% with the addition ¹³C-methanol and ¹³C-CO₂ (Fig. 4D). These results further demonstrated that MFORG-expressing P. pastoris could co-assimilate methanol and CO₂.

Adaptive laboratory evolution strategies have proven to be a very successful tool for improving cellular phenotypes. Following this approach with reverse engineering to find mutated genes associated with improved phenotypes is a valuable method^{34, 35, 36, 37}. To further improve the growth performance of PMORG09, we used ALE to improve its adaptation to a minimal medium with methanol as the sole carbon source. After 4 months of evolution, when compared with our initial strain, PMORG09, the growth rate of the evolved strain had improved by 30% (Supplementary Fig. 2). Subsequently, three evolved strains were isolated and their genomes were sequenced, with seven mutations correspondingly obtained (Supplementary Table 2). The mutated genes were restocked into PMORG09 and peroxisomal membrane protein (845T > G), involving in peroxisomal transmembrane transport, had a positive effect on improving growth rate of engineered P. pastoris on methanol.

Construction of synthetic S. cerevisiae growing solely on methanol with CO ₂ fixation

We used the synthetic MFORG pathway instead of the original XuMP cycle in P. pastoris to achieve co-assimilation of methanol and CO₂. To further extend the application of this synthetic methanol pathway, we chose to transplant the entire pathway into S. cerevisiae. Similarly, we integrated the methanol oxidation module into S. cerevisiae using a peroxisome-based compartmentalization strategy by fusion expression of the PTS1 signal peptide with AOX, FLD, and FGH genes. We then expressed MIS1, GCV1, GCV2, and GCV3 genes from P. pastoris to integrate the C1-compound co-assimilation module into the genome of S. cerevisiae (Fig. 5A). Lastly, the endogenous pyruvate synthesis module of the SHM1 gene was overexpressed. As a result, a synthetic S. cerevisiae strain, SMFORG1 was obtained. To characterize the growth of strain SMORG1 on methanol, we cultured it in minimal salt medium supplemented with methanol as the sole carbon source. The wild-type strain BY4741, which failed to grow on methanol, was used as a control (Fig. 5B). As the results indicate in Fig. 5B, the synthetic strain SMFORG1 was able to grow on methanol solely. Over the course of the 144-hour fermentation, SMFORG1 could grow to an OD₆₀₀ of 0.34 from an OD₆₀₀ of 0.2. During the entire fermentation process, 2.88 g/L methanol was consumed with a methanol consumption rate of 24 mg/L*h (Fig. 5C). These values are the highest levels reported for synthetic methylotrophic S. cerevisiae to date. There results indicated that expression of the synthetic MFORG pathway may convert S. cerevisiae to a synthetic methylotroph growing solely on methanol.

To validate incorporation of methanol and CO₂, in vivo ¹³C metabolic tracer assays were also performed. We cultured SMFORG1 in minimal salt medium supplemented with ¹³C-CO₂/¹²C-Methanol, ¹²C-CO₂/¹³C-Methanol, and ¹³C-CO₂/¹³C-Methanol respectively. As shown in Fig. 5D, 5E, and 5F, nine amino acids were detected. When the strain was grown with ¹²C-CO₂/¹³C-Methanol, all amino acids derived from central carbon metabolism were labeled by carbons from ¹³C-methanol (Fig. 5E). For the intermediates in the MFORG pathway, 38% and 23% of the Gly pool contained M + 1 and M + 2 labeling, respectively (Fig. 5E). Additionally, 45% of the Ser pool contained M + 1 labeling, 22% of the Ser pool contained M + 2 labeling, and 9% of Ser pool contained M + 3 labeling (Fig. 5E). The labeled fraction of the other amino acids is also over than 65% (Fig. 5E), indicating the methylotrophic ability of engineered MFORG-expressing S. cerevisiae. When feeding with ¹³C-CO₂/¹²C-Methanol, labeled carbons in these amino acids were also detected (Fig. 5D). These results identified that the synthetic methylotrophic S. cerevisiae SMFORF01 was capable of assimilating CO₂. The ability to co-assimilate methanol and CO₂ was further determined by a 13-C labeling approach. The further evidence that CO₂ and methanol were both incorporated were confirmed by labeling of amino acids in ¹³C-CO₂/¹³C-Methanol. The labeling fraction of Ala, Ser, and Thr was found in over 92% (Fig. 5F). For example, the labeling pool of Alaine reached 99% (Fig. 5F). These results demonstrated that the carbons from methanol and CO₂ passing through the MFORG pathway were used by central carbon metabolism to synthesize various metabolites. This further demonstrated that the methylotrophic S. cerevisiae strain SMFORG01 can indeed grow solely on methanol and also co-assimilate CO₂.

The ability of synthetic P. pastoris and S. cerevisiae to grow on formic acid solely

In the MFORG pathway, formic acid was one of the metabolic intermediates. When formic acid is used as a carbon source, the reducing power and energy may be generated from FDH in the formic acid oxidation module to support cell growth. Therefore, it was supposed that synthetic P. pastoris and S. cerevisiae developed above could also use formic acid as the sole carbon and energy source to support cell growth. To identify this ability, PMORG09 and SMORG01 were cultivated in minimum salt medium supplemented with formic acid as the sole carbon source. As illustrated in Fig. 6A and 6B, compared to the wild-type GS115, PMORG09 exhibited more favorable growth with a 105% improvement in formic acid consumption rate. The cells grew to an OD₆₀₀ of 0.35 from an OD₆₀₀ of 0.2, while no growth was observed in the wild-type GS115. Similarly, S. cerevisiae SMORG01 could also successfully grow in minimal salt medium with formic acid as the sole carbon and energy source and 1.8 g/L of formic acid was consumed. The OD₆₀₀ of SMORG01 was reduced to 0.29 from 0.2. However, due to the limited energy production from oxidation of formic acid to CO_2, both the synthetic P. pastoris and S. cerevisiae exhibited lower growth performance on formic acid than those grown with methanol as the sole carbon source. As CO₂ was an essential co-substrate of the MFORG pathway, the co-assimilation of CO₂ with formic acid utilization was anticipated in these synthetic P. pastoris and S. cerevisiae. Nonetheless, the ability of these synthetic yeasts to achieve CO₂ fixation using methanol or formic acid is positive and further broadens the utilization of C1-compounds by synthetic yeasts.

Biotransformation of methanol to lactic acid and 5-aminolevulinic acid using engineered P. pastoris and S. cerevisiae

To verify if the engineered P. pastoris and S. cerevisiae was capable of producing value-added chemicals from C1-compounds, lactic acid and 5-aminolevulinic acid production ability from methanol was selected as the proof-of-concept by further engineering the product pathway within our engineered P. pastoris PMORG09 and S. cerevisiae SMORG01. Lactate dehydrogenase from Lactococcus lactis, which catalyzed synthesis of lactate from pyruvate, was overexpressed in engineered PMORG09 and SMORG01 respectively (Fig. 7A). After 72-hours of growth in minimum salt medium with methanol as the sole carbon source, we successfully detected production of 73 mg/L lactic acid in engineered PMORG09-LDH, while none of the lactic acid was detected in engineered SMORG01-LDH (Fig. 7B).

To fully demonstrate the production performance of our synthetic methylotrophic yeasts, we chose the production of 5-aminolevulinic acid (ALA) as an example. ALA is an industrial fine chemical, and has important physiological functions in humans and other organisms, including acting as a substrate for heme biosynthesis³⁸. We overexpressed the 5-aminolevulinate synthase from Saccharomyces cerevisiae in both engineered PMORG09 and SMORG01. After 72-hours of growth in minimum salt medium with methanol as the sole carbon source, production of 5-aminolevulinic acid was successfully detected in PMORG09-HEM1 and SMORG01-HEM1, in which the titer was 0.71 mg/L and 1.67 mg/L, respectively. The product was analyzed by gas chromatography-time-of-flight mass spectrometry (GC–TOF-MS) which also identified the successful synthesis of 5-aminolevulinic acid (Supplementary Fig. 3). These results indicated that our synthetic methylotrophic yeasts could be used as hosts to produce value-added chemicals from C1 feedstock.

Previous studies have indicated significant achievements in developing the synthetic microorganisms capable of using multiple one carbon compounds in native or synthetic bacteria^{3, 16}. The efforts related to CO₂ fixation with methanol or formate as the energy source has been pursued in recent years. With native anaerobic methylotrophic-acetogenic bacterium, such as Butyribacterium methylotrophicum, the co-utilization of methanol and CO₂ to synthesize organic acids has been achieved^{39, 40}. Methylobacterium extorquens AM1, an aerobic methylotrophic bacterium, was converted for CO₂ fixation by expressing a heterologous CBB cycle with methanol to provide electrons⁴¹. E. coli, the model bacterium, has also been converted to act as a synthetic methylotroph or formatotroph with the ability to co-assimilate CO₂^{20, 22, 24, 41}. However, development of synthetic yeasts to efficiently utilize one carbon compounds has been limited to methanol bioconversion using native methylotropic P. pastoris. In this study, we designed a synthetic MFORG pathway by combining the methanol and formic acid oxidation processes with the reductive glycine pathway. The pathway begins with methanol, where formic acid is a metabolic intermediate, and CO₂ acts as an essential co-substrate to make it work. Therefore, this provides great potential to enable the strain to utilize a variety of C1-compounds.

Based on the synthetic MFORG pathway, the native methylotropic P. pastoris was successfully converted to be able to co-assimilate methanol and CO₂. Meanwhile, the methanol consumption rate and biomass yield of the final engineered P. pastoris PMORG09 was significantly higher than the widely-used wild type GS115 (Fig. 3D, Table. 1), indicating the great advantages of the synthetic MFORG pathway in improving methanol bioconversion by P. pastoris. When the MFORG pathway from P. pastoris was transplanted into S. cerevisiae, it was successfully converted into a full synthetic methylotroph grown on methanol for CO₂ fixation (Fig. 5B). High amounts of effort have been made for synthetic methanol assimilation in S. cerevisiae¹⁸. Most often, XuMP cycle from methylotrophic yeast, and the ribulose monophosphate (RuMP) cycle from methylotrophic bacteria have been employed²⁷. However, the growth of S. cerevisiae using methanol as the sole carbon source is still challenged. A co-substrate such as xylose, yeast extract or amino acids have often been required to support growth on methanol^{42, 43, 44} (Supplementary Table 3). The synthetic MFORG pathway could successfully support S. cerevisiae to grow solely on methanol without extra metabolic modifications. Furthermore, the methanol consumption rate of the synthetic S. cerevisiae using the MFORG pathway reached 0.75 mM/L*h (Fig. 5C), the highest methanol assimilation level in a methylotrophic S. cerevisiae reported to date.

Notably, the synthetic P. pastoris and S. cerevisiae with the MFORG pathway could also grow on formic acid solely (Fig. 6). The synthetic formatotroph has been widely established in E. coli, especially co-assimilation of formic acid and CO₂ based on use of the rGly pathway^{9, 20}. The feasibility of the rGly pathway was tested in an engineered S. cerevisiae auxotrophic for glycine and formate-dependent growth was achieved³¹. However, the growth of yeasts with formic acid as the sole carbon and energy source had never been demonstrated. Based on the synthetic MFORG, P. pastoris and S. cerevisiae could be engineered to use both methanol and formic acid as carbon sources, indicating the advantages of the synthetic MFORG in broadening the type of one carbon compound, as well as the flexibility of yeast as promising hosts for bioconversion of multiple one carbon compounds. In addition, when cells were cultured with formic acid, the final biomass of synthetic P. pastoris and S. cerevisiae was lower than those with methanol (Fig. 6A). When we knocked out the formic acid oxidation module, aiming to decrease CO₂ release in methanol medium, we found that the growth of the strain was significantly decreased (Fig. 2B). These results suggested that the efficient energy supply was still one of important factors for C1-compound utilization in synthetic strains. The further balance of energy and carbon metabolism, or the introduction of another energy supply system, such as electricity or photosystem^{45, 46, 47}, may be an alternative approach to increase the bioconversion efficiency of C1-compounds.

The synthetic P. pastoris and S. cerevisiae in our study could also assimilate CO₂ simultaneously due to the presence of the rGly pathway (Fig. 4B and Fig. 5D) ⁴⁸. Presently, the highest specific CO₂-assimilation rate in yeast was 0.03 g/g/h by an engineered P. pastoris with a methanol co-feed¹⁶. In the synthetic MFORG pathway, 1 mol of CO₂ is fixed with 2 mol of methanol or formic acid consumption theoretically. Based upon the methanol and formic acid consumption rates, the synthetic P. pastoris enabled a CO₂ fixation rate of 0.036 g/L/h (0.29 g/g_DCW/h) with methanol co-feed and a CO₂ fixation rate of 0.019 g/L/h (0.27 g/g_DCW/h) with formic acid co-feed, while the CO₂ fixation rate of 0.017 g/L/h (0.25g/g_DCW/h) and 0.010 g/L/h (0.2 g/gDCW/h) were achieved by synthetic S. cerevisiae with methanol and formic acid co-feed respectively. In addition, the growth of synthetic yeasts was not independent of the supply of high CO₂ concentration as reported in rGly-equipped E. coli^{20, 21}. This suggested the potential of synthetic P. pastoris and S. cerevisiae in the direct utilization of atmospheric CO₂ or the recycling of CO₂ from methanol or formic acid oxidation.

Yeasts have been proven to be a promising chassis for high production of various proteins and value-added chemicals due to their high gene expression level, post-translational modification system, as well as high tolerance to extreme industrial fermentation conditions^{25, 49, 50}. Engineering C1 assimilating yeasts was an important track for developing methanol-based biomanufacturing¹⁸.From the synthetic methylotrophic and formatotrophic developed in our study, value-added chemicals, especially those derived from the MFORG pathway intermediates, could be easily engineered for production from C1-compounds (e.g., methanol, formic acid, and CO₂). Due to the safety of yeast in producing foods or medicine²⁵, a new route for the production of some important products is present. For example, in combination with a high-density fermentation strategy, the microbial protein production from liquid C1-compounds and CO₂ could be achieved. Therefore, the strategies and synthetic yeasts developed in this study provide novel routes and host systems for further advances in C1 biorefining toward the production of chemicals and materials from C1-compounds, and is also benefit for mitigation of atmospheric CO₂.

Strains and genes. All primers and strains used in this study are listed in Table Supplementary Table 4 and Supplementary Table 5. E. coli Trans1-T1 was used as a gene cloning host and base strain for developing the engineered formatotrophic strain. Genes AOX1, FLD, FGH, MIS1, GCV1, GCV2, GCV3, and SHM1 required for methanol assimilation were from P. pastoris. The gene encoding lactate dehydrogenase (LDH), which catalyzes the synthesis of lactate from pyruvate, was from Lactococcus lactis. The gene encoding 5-aminolevulinate synthase (HEM1) was from S. cerevisiae, and the enzyme which catalyzes the synthesis of 5-aminolevulinic acid from glycine. S. cerevisiae BY4741 was used as the background strain for all genetic manipulation.

Medium composition and Cultivation. Yeast Extract Peptone Dextrose (YPD) medium and minimal salt medium (pH 7.0) were used in this study. The YPD medium contained (per liter) 20 g tryptone, 10 g yeast extract and 20 g glucose. The minimal salt medium contained (per liter) 6.8 g Na₂HPO₄·7H₂O, 3 g KH₂PO₄, 0.5 g NaCl, 2 g NH₄Cl, 4.2 g NaHCO₃, 0.8 g MgSO₄·7H₂O, 0.04g Histidine, 4*10^− 4g Biotin, and 10mL methanol. Cells were inoculated into 5 ml liquid YPD medium and grown for 12 hours. For cultivation using methanol alone, the precultured cells (OD₆₀₀ of 5–6) were washed once with fresh minimal salt medium to remove all other nutrients, resuspended in 1.5 mL fresh minimal salt medium and added to a flask containing minimal salt medium supplemented with 10 mL x L^− 1 methanol to give an initial OD₆₀₀ of 0.2. Cells were grown at 200 rpm and 30°C using a shaker.

Deletion of genes from genomic DNA. Deletion of the DAS gene from the genome of the GS115 strain was carried out following the homologous recombination method. Construction of the upper and lower 1000bp homologous arms of the DAS1 and DAS2 gene into plasmid pET-28a was conducted by overlap PCR. Plasmids were electroporated P. pastoris, and positive transformants were selected in MD medium.

Determination of ¹³ C‑labeled metabolites. To determine the levels of ¹³C-labeled intracellular metabolites, assimilation of methanol by recombinant yeast strains was monitored by a ¹³C-methanol labeling assay. The recombinant strains were cultured in YPD medium for 10–12 hours. The cells were then transferred to minimal salt medium containing 300 mM ¹³C-methanol (Sigma-Aldrich). After 144 hours of fermentation, cells were collected and pretreated as follows. The supernatant of the metabolite extraction (800 µL) was transferred to a 1.5 mL tube and lyophilized at low temperature. The dried extract was then reconstituted in 300 µL of acetonitrile: H₂O (1:1, v/v), sonicated for 10 min, and centrifuged at 10,000 × g and 4°C for 15 min to remove insoluble debris. The supernatant was transferred to an HPLC vial and stored at -80°C prior to LC-MS/MS analysis. For each sample, three replicates were performed. We used the same method to add ¹³C-NaHCO₃ to monitor assimilation of CO₂ by the recombinant yeast strain.

Analytical methods. Cell growth was determined through measurement of OD₆₀₀ using a Beckman Coulter DU370 spectrophotometer. Methanol concentrations and formic acid concentration were measured using an Agilent 1290 Infinity System (Santa Clara, CA, USA) equipped with an Aminex HPX-87H column. LC–MS/MS analyses were performed using an UHPLC system (1290, Agilent Technologies) equipped with a UPLC BEH Amide column (1.7 µm, 2.1*100 mm, Waters) coupled to Triple TOF 6600 (Q-TOF, AB Sciex). The mobile phase consisted of 25 mM NH₄OAc and 25 mM NH₄OH in water (pH = 9.75) (A), and acetonitrile (B) was carried with elution gradient as follows: 0 min-95% B, 0.5 min-95% B, 7 min-65% B, 8 min-40% B, 9 min-40% B, 9.1 min-95%, and B, 12 min-95% B, which was delivered at a rate of 0.5 mL/min. The injection volume was 1 µL. The Triple TOF mass spectrometer was used to assess its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during an LC/MS experiment. ESI source conditions were set as follows: ion source gas 1 as 60, ion source gas 2 as 60, curtain gas as 35, source temperature 550°C, ion spray voltage floating (ISVF) 5500 V or − 4500 V in positive or negative modes, respectively.

Subcellular location. To confirm targeted protein localizations, a green fluorescent protein (GFP) carrying a peroxisomal signal (PTS1) was fused to C-termini of fgh1, and a red fluorescent protein (RFP) carrying PTS1 was fused to N-termini of fld. The recombinant plasmid was transformed into P. pastoris. Cells were grown in minimal media for 12–16 hours at 30°C, shaken at 200 rpm. The cell cultures were observed by microscope (Leica Microsystems CMS GmbH)

Lactic acid measurement in strain. Lactic acid concentration was measured using an Agilent 1290 Infinity System (Santa Clara, CA, USA) equipped with an Aminex HPX-87H column. Engineered strains were pre-cultured in 5-mL aliquots in YPD medium overnight and used to inoculate 50 mL minimal salt medium (10mL x L^− 1 methanol). The cultures were grown at 30°C and shaken at 220 rpm in an orbital shaking incubator. After fermentation, the supernatant of the fermentation broth was centrifuged and tested for lactic acid content in an Agilent 1290 Infinity System.

5- aminolevulinic acid measurement in strain. Engineered strains were pre-cultured in 5-mL aliquots of YPD medium overnight and used to inoculate 50 mL minimal salt medium (10mL x L^− 1 methanol) in 500 mL. Cultures were grown at 30°C and shaken at 220 rpm in an orbital shaking incubator. At the end of fermentation, 200 µL of supernatant was lyophilized and derivatized with N, O-Bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane and methoxyamine hydrochloride. Next, 5-Aminolevulinic acid was detected by LC-MS/MS analysis.

Acknowledgements

This work was supported by the Development Program of China (2018YFA0901500) and National Key Research, the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (XTD2218).

Author contributions

Kequan Chen and Xin Wang conceived and coordinated the project; Kequan Chen and Xin Wang designed research; Yuanke Guo performed experiments; Yuanke Guo, Rui Zhang, Jing Wang, Ruirui Qin and Jiao Feng analyzed data; Xin Wang and Yuanke Guo wrote the manuscript and all authors approved the final manuscript.

Competing interests

The authors declare that they have no conflicts of interest.

Data availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request. Genome sequencing reads of mutant strains are available at NCBI Bio Project SAMN33688472. The NCBI reference sequence and other information of all native and heterologous sequences are shown in Supplementary Table 6. Materials will be made available for non-profit research under a material-transfer agreement upon reasonable request to the corresponding author.

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Engineering Yeasts to Grow Solely on Methanol or Formic acid coupled with CO₂ fixation

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The synthetic methanol and formic acid oxidation-reductive glycine pathway

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