Metabolic Engineering of Saccharomyces cerevisiae for Conversion of Formate and Acetate into Free Fatty Acids

Abstract

The ever-increasing global energy demand, juxtaposed with critical concerns about greenhouse gas emissions, emphatically underscores the urgency to pivot toward sustainable and eco-friendly energy alternatives. Tapping into microbial metabolism for clean energy generation stands out as a particularly promising avenue in this endeavor. Given this backdrop, we delved deeply into the metabolic engineering potential of Saccharomyces cerevisiae, thereby aiming for the bioconversion of formate and acetate—both CO₂ derivatives—into free fatty acids (FFAs) as precursors for biofuel production. Our study not only elucidated the metabolic pathways within S. cerevisiae that are tailored for efficient formate and acetate utilization but also shone a light on the meticulous optimization strategies that amplify FFA synthesis. The engineered strains, under refined conditions, exhibited up to an 8-fold increase in an FFA titer, thus reaching a production level of 6.6 g/L, which showcases the potential of microbial metabolism in clean energy generation. Our findings offer a promising step toward harnessing microbial metabolism for sustainable energy production, thereby bridging the gap between waste carbon utilization and greener fuel alternatives.

1. Introduction

Greenhouse gas emissions, i.e., those primarily resulting from human activities, have become a significant concern in the context of global climate change. These emissions, which comprise a range of gases including carbon dioxide (CO₂), methane (CH₄), nitrous oxide (N₂O), and fluorinated gases, are directly associated with multiple and multifaceted environmental challenges [1]. Also, they have led to a series of environmental consequences, such as rising sea levels and unpredictable weather events [2]. In recognizing the severity of the issue, the scientific community has been tirelessly working toward finding sustainable solutions. The primary focus of recent research and development efforts lies in the realm of alternative energy sources and carbon utilization technologies. One such promising avenue has been the utilization of low-cost biomass, which presents an environmentally friendly alternative to fossil fuels [3]. Specific emphasis has been given to the exploitation of CO₂ and its photoelectrochemically derived molecules, such as formic acid [4,5,6], methanol [7,8,9], acetic acid [10,11,12,13], etc. Among the aforementioned compounds, acetic acid holds a special place. The microbial conversion of acetic acid into value-added products or energy has made considerable strides in recent years. Several microorganisms have been engineered for this purpose. Escherichia coli, a common bacterium present in the intestines of warm-blooded organisms, has shown the potential to efficiently convert acetic acid into useful chemicals [14,15,16]. Similarly, Corynebacterium glutamicum, known for its role in the production of amino acids, has also demonstrated capabilities in processing acetic acid effectively [17,18]. Another notable microorganism in this context is Yarrowia lipolytica, a type of yeast which has shown promise in high-yield transformations of acetic acid [19,20].

Acetic acid, while promising as a low-cost biomass for microbial conversion, brings forth certain challenges. One significant drawback is its demand for an ample amount of reducing power in the microbial conversion process. These challenges highlight the necessity for an efficient energy carrier system. One-carbon chemical energy carrier transfer systems have emerged as a potential solution. Within this system, specific carriers such as formate, methanol, H₂, and CO can be synthesized utilizing relatively low-drive voltages [21]. Of these, H₂ and CO, while abundant, come with inherent challenges due to their flammability and possible safety concerns. On the other hand, formate has stood out with several inherent advantages. Its non-volatility ensures safer handling and storage [22,23]. Methanol, another promising candidate, has been noted for its superior attributes, such as its high solubility and optimal redox potentials [24,25]. What adds to their appeal is that both formate and methanol do not require external electron acceptors, thus positioning them as front runners for one-carbon chemical energy carriers.

In a broader perspective, free fatty acids (FFAs) play an indispensable role in various industries and biological processes. These molecules are essential components of cell membranes and function as crucial energy storage units. Their industrial applications are vast and varied, from being key ingredients in biodiesel production to serving as integral components in cosmetic formulations [26]. As our world steadily drifts toward more sustainable alternatives, recent breakthroughs in metabolic engineering and synthetic biology have paved the way for the microbial synthesis of FFAs. Microorganisms such as E. coli [27,28,29], S. cerevisiae [30,31,32], and Ogataea polymorpha [25,33,34] have been harnessed for this purpose. This bio-based approach to producing FFAs not only presents an environmentally friendly alternative to traditional petroleum-derived methods but has also underscored a paradigm shift toward a more sustainable type of bioeconomy.

In our endeavor, we targeted the yeast Saccharomyces cerevisiae, a model organism with a rich history in biotechnological applications, which—in the field of acetate conversion—still remains relatively untapped. Our goal was to break new ground by pioneering a unique method for the simultaneous conversion of formate and acetate into FFAs, as illustrated in Figure 1. At the heart of this innovative process is acetate, which serves a pivotal role by acting as the primary backbone. It provides the necessary carbon skeleton, which is quintessential for cellular growth and the ensuing production processes. Parallel to this, formate stepped in to fulfill a distinct yet equally vital function. By catering to the system’s energy demands, the formate complemented the role of the acetate, thus ensuring an optimal balance in the conversion process. The collaboration between these two substrates was not just additive; it was synergistic. Their combined interactions formed a powerful partnership that made the efficient synthesis of free fatty acids not just possible but also highly effective.

But the implications of our research extend far beyond the laboratory. By harnessing the potential of such readily available and affordable biomasses, we can provide more than just a short-term remedy for our current environmental challenges. We are laying a solid and adaptable foundation for a myriad of future endeavors. This foundation will center on the creative utilization and transformation of CO₂ and its derivatives, thereby tapping into a vast reservoir of opportunities through which to mitigate the effects of climate change.

Thus, we aim to pioneer a method in Saccharomyces cerevisiae, where acetate conversion research is still nascent, for the simultaneous transformation of formate and acetate into FFAs (Figure 1). In this process, acetate serves as the primary backbone, thereby offering the carbon skeleton essential for cellular growth and production. Concurrently, the formate catered to the energy requirements. Their synergistic interaction facilitated the efficient synthesis of free fatty acids. Harnessing such affordable biomasses was not merely an immediate solution as they also lay a robust foundation for future endeavors that are centered on the utilization and transformation of CO₂ and its derivatives. This holistic approach promises a more sustainable and integrated bioeconomy, thus going some way to satisfy the global call for greener solutions.

2. Materials and Methods

2.1. Strain and Plasmid Constructions

The plasmids, primers, strains, and exogenous genes employed in this study are comprehensively detailed in Tables S1–S4. The yeast background strain employed for further engineering in this study was YJZ08, a derivative of the S. cerevisiae strain CEN.PK113-11C [35].

The gene sequences of acyl-CoA synthetase (ACS) from various hosts were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/ (accessed on 17 September 2023). Subsequently, Sangon Biotech (Shanghai, China) conducted codon optimization and gene synthesis for these sequences. The formate transformation (formate dehydrogenase, FDH) plasmids were derived from the foundational research conducted within our laboratory [23]. Gene fragments were acquired via PCR, and were then subsequently integrated into the vector pIYC04 via restriction enzyme digestion and ligation. NotI and XhoI enzymes were specifically employed for the restriction enzyme digestion process. The fidelity of all gene constructs was validated via sequencing. Competent cells of S. cerevisiae were prepared utilizing the ZYMO Frozen-EZ Yeast Transformation II Kit (Zymo Research, Irvine, CA, USA) for transformation purposes.

2.2. Fermentation Conditions

The genetically modified strains were cultivated on a synthetic complete medium lacking uracil and/or histidine. All yeast strains were cultured under consistent conditions at 30 °C and 200 rpm in shake flasks. Detailed information regarding the specific reagents employed in this study, along with their concentrations, can be referenced in previously published articles [23,36].

Shake flask fermentations were conducted in a defined minimal medium containing 20 g/L of glucose, 10 g/L of sodium acetate, 5 g/L of (NH₄)₂SO₄, 5 g/L of sodium formate, 14.4 g/L of KH₂PO₄, and 0.5 g/L of MgSO₄·7H₂O. This medium was further supplemented with trace metals and vitamin solutions. Additionally, when necessary, 40 mg/L of histidine and/or 40 mg/L of uracil were added to the medium.

2.3. Fed-Batch Fermentation

Fed-batch fermentations for the production of free fatty acids (FFAs) were carried out in 5.0-L bioreactors (with an initial working volume of 2 L) using a Biotech-5JG System (BXBIO, Shanghai, China). The initial batch fermentation employed a minimal medium comprising 5 g/L of (NH₄)₂SO₄, 14.4 g/L of KH₂PO₄, 0.5 g/L of MgSO₄∙7H₂O, and 20 g/L of glucose, along with trace metals and vitamin solutions. The fermentation conditions were maintained at a temperature of 30 °C with the agitation speed set at 400 rpm. To maintain a stable pH level, 2 M of H₂SO₄ was employed, thereby ensuring that the pH was consistently maintained at 5.6 throughout the fermentation process. Following a 24 h preincubation period, 5 or 10 g/L of acetate and 5 g/L of formate were introduced. These compounds were replenished once they were completely consumed in the fermentation process. After fermentation, 20% (v/v) ethyl acetate was introduced for the extraction of the fatty acids. The entire fermentation broth was resuspended in the original fermentation medium to quantify the total production of the FFAs [25]. Experiments were conducted in triplicate.

2.4. Analytical Methods

Biomass was quantified by measuring the optical density at 600 nm (OD₆₀₀) using an EU-2600 visible spectrophotometer (Shanghai Onlab Instruments, Shanghai, China).

Extracellular metabolites, including glucose, acetate, formate, and ethanol, were quantified using high-performance liquid chromatography (HPLC) with a Shimadzu LC-20AT system (Kyoto, Japan). An HPLC was equipped with RID and UV detectors, which was then set at 210 nm and used to measure extracellular metabolites. The system utilized an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) at 65 °C, with 5 mM of H₂SO₄ as the eluent and a flow rate set at 0.6 mL/min. For each analysis, the injection volume was maintained at 10 μL.

Samples of the FFAs were collected at 96 h for analysis via gas chromatography–mass spectrometry (GC-MS, QP2020, Shimadzu, Kyoto, Japan), which was utilized following the derivatization method described in our recent publication [13]. The process began with transferring 200 μL of the cell culture to a glass vial. This was immediately followed by the addition of 10 μL of 40% tetrabutylammonium hydroxide and 200 μL of the methylation reagent. After shaking the mixture for 30 min, it was centrifuged at 5000× g for 3 min. Subsequently, 100 μL of the supernatant, containing the methyl esters, was transferred to a GC vial. This sample was then left to evaporate for 6 h and reconstituted in 100 μL of hexane. The analysis on the GC-MS (QP2020, Shimadzu, Japan) utilized a DB-5MS column (30 m × 0.250 mm × 0.25 μm, Agilent). Our temperature protocol included an initial hold at 40 °C for 2 min; this was increased at a rate of 5 °C/min to 130 °C, followed by a ramp of 10 °C/min to 280 °C, which was then sustained for 3 min. The temperatures for the inlet, mass transfer line, and ion source were maintained at 280 °C, 300 °C, and 230 °C, respectively. A constant carrier gas flow rate of 3.0 mL/min was used, with an injection volume of 1 μL. The data were captured in full scan mode (50–650 m/z) and then processed using GCMS Solution 4.4 software.

2.5. Statistics

The data are presented as the average ± standard deviation, which was based on three separate biological samples (n = 3). A two-tailed t-test was used for statistical evaluation using Microsoft Excel as the statistical program.

3. Results and Discussion

3.1. Metabolic Engineering Enables Acetate Conversion in Yeast

In the pursuit of harnessing microbial mechanisms for acetate transformation, we undertook a comprehensive approach by turning our attention to the acetyl-CoA synthetase (ACS) pathway. This pivotal enzyme, responsible for the activation of acetate, is a cornerstone in the metabolic pathways of many organisms [19].

Therefore, we first screened various ACSs. Based on previous reports, ACS1 (ScACS1, 1.10 U/mg-protein) and ACS2 (ScACS2, 0.34 U/mg-protein) from Saccharomyces cerevisiae demonstrated considerable enzymatic activity [37]. Additionally, the ACS mutant (SeACS^L641P) from Salmonella enterica also exhibited high enzymatic activity (47.0 mU/mg-protein) [38]. Moreover, due to the close phylogenetic relationship between Yarrowia lipolytica and Saccharomyces cerevisiae, ACS1 (YlACS1) and ACS2 (YlACS2) from Yarrowia lipolytica were also selected for the conversion of acetate into Saccharomyces cerevisiae. Our initial investigations focused on expressing the ACS from various species in the YJZ08 strain. YJZ08 is not just any random strain; it has already marked its niche in the realm of microbial metabolism with its commendable capability in FFA production [35]. Thus, embedding ACS into this strain presented a compelling synergy, wherein it was poised to enhance acetate transformation.

Once the strains were engineered, the test of their capability was initiated via co-substrate fermentation, which was achieved by employing both glucose and acetate. Within a span of 24 h, it was observed that all strains had completely metabolized the glucose, as illustrated in Figure 2A. However, it was the acetate metabolism that truly piqued our interest. Both our control strain, YJZ08, and its ACS-augmented counterparts showcased an appetite for acetate. In drawing insights from Figure 2B, a side-by-side comparison unfurled a stark contrast. The control strain, YJZ08, had a moderate appetite, whereby it consumed 4.52 g/L of acetate over a span of 96 h. In stark contrast stood the experimental strain WZ04, which voraciously utilized acetate, thereby registering a consumption of 9.12 g/L. This was not a mere incremental increase—it effectively doubled the rate at which the control strain metabolized acetate.

Further analysis, as shown in Figure 2C, revealed that the introduction of the ACS led to a significant enhancement in the production of FFAs across the tested strains. Among them, WZ04 and WZ05 outperformed, registering a nearly 100% increase in FFA production, thus reaching levels of 642.30 mg/L and 640.57 mg/L, respectively. This suggested that, with the strategic introduction of the ACS, we not only optimized acetate conversion but also significantly elevated FFA production in the strains. Taking into account both the acetate conversion rate and the production of FFAs, the WZ04 strain was selected for further research.

3.2. Introducing the Formate Pathway for Energy Supply: A Promising Approach

In acknowledging the intricate and energy-demanding nature of acetate conversion and FFA production, our investigative lens pivoted to one of the most fundamental aspects of microbial metabolism: the source and provision of cellular energy. This shift in focus became imperative, not just for the seamless continuation of these processes but also for optimizing their yield and efficiency.

Formate, a derivative of CO₂, emerged as a potent candidate for this role. Its ubiquity, thanks to the pervasive presence of CO₂, combined with its favorable low reduction potential, makes it an attractive substrate. Beyond its inherent characteristics, formate holds larger promise in the realm of microbial metabolism. Its potential as an energy substrate for microbial utilization is both profound and underexplored, thus marking a horizon teeming with possibilities. Drawing inspiration from our foundational research, we had previously stumbled upon a revelation: the process of formate dissimilation. Under the catalytic action of formate dehydrogenase (FDH), formate can be converted into CO₂. This process is accompanied by the generation of NAD(P)H, which is beneficial for the regeneration of intracellular cofactors, thereby providing an ample energy source for the synthesis of the product—free fatty acids (FFAs). This pathway, as elaborated in our earlier findings [23], presented a promising gateway for ensuring a steady and efficient cellular energy supply.

Given the promise formate holds, the next logical step in our exploration was the enzyme responsible for its metabolism: FDH. Our hypothesis postulated that by strategically introducing various types of FDH into our system, we could fine-tune and, perhaps, amplify the energy derivation process from formate. This was not only about validating the efficacy of these enzymes but was also a step toward deciphering which FDH would best complement our system for optimized energy provision.

Given our prior understanding and the intriguing promise showcased by WZ04 in acetate conversion, our next strategic move was set in stone: we would employ the optimal WZ04 strain (which was now re-engineered to express the formate dehydrogenase). Our goal was straightforward yet profound—observe and understand its growth dynamics and production capabilities, especially when operating under the energy auspices of formate.

In a well-calibrated experiment, we introduced external formate, envisaging it as a supplementary energy for our engineered strains. The outcomes, as they began unfolding, were nothing short of exhilarating. Figure 3A, with its neatly plotted data points, told a story of microbial prowess: our strains were not just metabolizing formate.

However, formate was not the only substrate utilized under microbial conditions utilized. Glucose, an omnipresent and preferred carbon source for many microbes, vanished completely, a phenomenon that is visually captured in Figure S1. Acetate, the central theme of our research and the more challenging substrate was not left untouched either. As Figure 3B elucidates, it, too, was entirely consumed, thus hinting at the metabolic flexibility and efficiency of our engineered strain.

Interestingly, when we observed the consumption of formate (Figure 3A) and acetate (Figure 3B) simultaneously, we found that the formate consumption rate was higher during the period of approximately 50–70 h, but the assimilation of acetate suddenly stopped during this period. Surprisingly, formate consumption abruptly ceased afterward, and the acetate assimilation resumed again with a higher acetate consumption rate. We hypothesized that, during the 50–70 h period, the cells urgently required a certain number of cofactors to drive further acetate conversion; as such, during this stage, the cell metabolism shifted to formate metabolism to provide sufficient cofactors. Therefore, there was a noticeable conversion of formate; meanwhile, the acetate could not continue its high-efficiency conversion due to a lack of sufficient driving force. After 70 h, the cells accumulated a large number of cofactors from the conversion process of formate, which efficiently promoted the conversion of acetate. Hence, the conversion of acetate continued further; meanwhile, there was no significant change in the formate. However, we thought that the formate was still continuously being converted as the conversion process of acetate might produce formate; as such, the total amount of formate appeared unchanged. This was a particularly interesting discovery, and in future research on the conversion of formate and acetate, we intend to delve deeper into their internal metabolic regulation mechanisms.

But where things became genuinely captivating was in the domain of FFA production. Our hybrid strain, christened as WZF04 for clarity, did not just keep pace with its predecessors—it obliterated their records. Clocking in at a staggering 822.14 mg/L, it not only showcased a 27.99% productivity jump over its ACS-only counterpart, WZ04, but it also left the benchmark strain, YJZ08, trailing with a 161.17% production surge, as delineated in Figure 3C.

Intriguingly, as shown in Figure 3C, there was another revelation. Beyond the impressive FFA production numbers, there was a marked acceleration in the growth kinetics of the strains that were fortified with formate dehydrogenase and provided with exogenous formate. This was not just a footnote; it was a monumental observation that revealed the potential synergies between FDH expression and formate availability in driving microbial growth.

In wrapping up our observations, one thing became crystal clear: formate, with its untapped potential, is poised to become a linchpin in microbial metabolic engineering. The preliminary results here are not just a testament to the promise of integrating formate conversion pathways but also heralding a vibrant frontier for future investigations, thus potentially reshaping our approach to sustainable microbial production systems.

To provide a comprehensive understanding of our strains’ metabolic prowess, we embarked on a series of tests sans the external addition of formate. This was instrumental in discerning the true metabolic flexibility and innate capabilities of our FDH-expressing strains when operating without the crutch of supplementary energy.

The results were illuminating. Figure 4A paints a vivid picture of glucose’s fate, with the sugar being completely metabolized within the confines of a day. This rapid consumption was indicative of the metabolic hunger and efficiency of our strains. More interestingly, with the introduction of FDH, a significant acceleration in acetate metabolism was witnessed. As showcased in Figure 4B, all of the strains achieved full acetate utilization within 96 h. The standout performer, however, was the WZF06 strain, which consumed acetate at a blistering pace of 95.94 mg/L/h—a rate that is indicative of its metabolic prowess.

Transitioning our gaze to FFA production, FDH’s role became even more evident. Even in the absence of formate supplementation, the FDH-expressing strains augmented FFA production, thus suggesting an intrinsic metabolic benefit that was derived from FDH expression (Figure 4C). WZF06 clocked the highest FFA production, registering a commendable 643.31 mg/L. Via poring over the supplementary data (Figure S2), we found that the strains could generate minimal amounts of formate intrinsically during glucose and acetate metabolism. This internally produced formate could potentially be channeled to provide some degree of energy via the dissimilation pathway, further bolstering the metabolic efficiency of these strains.

In piecing together the narrative from our extensive evaluations, one fact stands tall: the WZF04 strain with its unparalleled efficiency in acetate conversion and FFA production in the presence of external formate. Its consistent and robust performance makes it the paragon for formate and acetate conversion. Drawing from these revelations, as well as from our overarching research objectives, we zeroed in on the WZF04 strain as the optimal candidate for our scaled-up fermentation endeavors. This choice sets the stage for further explorations and validations in the grand tapestry of sustainable microbial production systems.

3.3. Scaling up Fermentation: Achievements in a 5 L Fermenter

In our quest to robustly elucidate the production power of our preferred strain, WZF04, we embarked on a scaled-up fermentation journey. Recognizing that consistent nutrient availability is the linchpin of efficient microbial growth, we pioneered a continuous glucose supplementation strategy. Keeping a vigilant eye on substrate levels, every time there was a near-depletion of formate and acetate, we promptly reintroduced 20 g/L of glucose, along with 10 g/L of acetate and 5 g/L of formate. This cyclical substrate replenishment approach is documented in Figure 5A.

However, the endgame results were a tad unanticipated. The FFA yield, at the conclusion of the scaled-up fermentation, was a modest 2.4 g/L. Pondering the intricacies of our substrate concentration and regimen, a few theories emerged. One plausible explanation leans toward substrate interference. It is conceivable that, in the tug of war for cellular resources, the substrates might jostle for priority. This tussle can skew the cells’ focus, thus making them lean more toward self-replication and survival rather than channeling energy for FFA production. This theory gained more traction when we observed a dual-consumption lag, wherein both glucose and acetate were not optimally utilized, thus hinting at a potential substrate rivalry or even a form of inhibition. Our data captured this phenomenon, as evidenced by the subpar cell growth trends showcased in Figure 5A.

To further dissect this conundrum and validate WZF04′s innate capabilities, we decided to strip the environment down to its basics. Starting with an initial glucose injection of 20 g/L, we patiently waited for its complete assimilation before commencing a steady flow of just formate and acetate. This minimalistic approach bore fruit. With the absence of glucose post its complete utilization, the formate and acetate lived in harmony, whereby the cells efficiently metabolized both concurrently. The final crescendo was impressive: the optical density soared to an OD₆₀₀ of 40, culminating in a robust FFA yield of 5.0 g/L. When ignoring the initial addition of 20 g/L of glucose, the yield was 0.086 g/g of acetate, as visualized in Figure 5B. This experimental detour underscored the significance of substrate balance and its intricate interplay in optimizing microbial fermentation. It beckoned a deeper dive into the metabolic pathways and cellular priorities to harness the true potential of the microbial strains in sustainable production processes.

During the intricate ballet of the fermentation journey, we noticed a peculiar trend: the aggressive consumption of formate and acetate was accompanied by significant use of an acidic solution to stabilize the pH. This constant juggling act to maintain a pH equilibrium poses a myriad of challenges. The steady addition of an acidic solution could instigate an environment that is less than ideal for cell proliferation. Acidic environments can alter the cellular membrane’s integrity, impede nutrient uptake, and even disrupt critical metabolic pathways. This potentially hampers both cellular growth and the subsequent production of desired compounds.

Moreover, the tale of acetate’s role in this complex fermentation narrative warranted a deeper dive. Acetate, in essence, is a double-edged sword. On the one side, it is a crucial substrate, driving the very reactions we aim to harness. However, on the other side, when introduced in higher concentrations, it can act as a metabolic disruptor. Cells, when suddenly bombarded with elevated acetate levels, can experience osmotic pressure changes, enzymatic activity fluctuations, and even a shift in their energy metabolism. This “metabolic shock” can, in turn, slow down growth rates, reduce biomass, and even cause cellular stress responses, thus impeding the efficient conversion of substrates to products.

Given these multifaceted challenges, it was imperative to revisit our fermentation playbook. We embarked on a mission to fine-tune the acetate addition protocol. Recognizing that moderation might be the key, the quantity of acetate infused during the fermentation experiments was meticulously recalibrated. By optimizing the acetate concentration, we aimed to strike a harmonious balance wherein the cells could thrive, efficiently metabolize the available substrates, and robustly produce the desired output without being overwhelmed by external stresses. This exercise underscored the nuances of microbial fermentation and the importance of a holistic understanding of the cellular environment for optimal outcomes.

With an intent to optimize and derive better efficiency, we strategically recalibrated our approach by tempering the acetate addition to a moderated level of 5 g/L. Riding on the premise of past experiences, two distinct strategies were meticulously crafted for this trial. The first strategy revolved around a continuous, pulsatile supplementation of glucose, which was applied precisely at a concentration of 20 g/L, as visualized in Figure 5C. The second strategy, as depicted in Figure 5D, was a tad more conservative. Here, glucose was judiciously added only at the inception of the fermentation process, thereby allowing the system to evolve organically thereafter.

The results from these adjustments were illuminating. In terms of optical density (OD₆₀₀), a metric indicative of cell growth and density, the values soared to approximately 49.3 in the first strategy (Figure 5C) and to an even more impressive ~56.5 in the second (Figure 5D). These figures were not just mere numbers but a testament to the thriving microbial milieu, which was optimized to perform at its peak.

The story becomes even more intriguing when we cast our gaze upon the FFA yield. In the realm of fermentation, yield is the gold standard—it is a quantifiable measure of efficiency and effectiveness. In both strategic cases, the FFA yield made a substantial leap, cresting at 8.0 g/L for the continuously supplemented strategy (Figure 5C) and 6.6 g/L for the initial addition approach (Figure 5D). Ignoring the initial addition of 20 g/L of glucose, when continuously feeding 5 g/L of formate instead of the continuous supply of 10 g/L of formate, the yield reached 0.110 g/g of acetate, thus representing an increase of 27.90% in productivity (Figure 5D and

Table 1. Comparison of fuels and chemicals production by C1/C2 substrate conversion.

Host	C1/C2 Substrate	Co-Substrates	Products	Titer (g L−1)	Ref.
H. bluephagenesis	Acetate	Glucose	Mevalonate	121	[39]
E. coli	Acetate	---	Succinate	7.29	[40]
	Formate and CO2	Glucose	Malate	18.6	[41]
	Acetate	Glycerol	Ethanol	~4	[14]
Y. lipolytica	Acetate	Glucose	Fatty alcohols	0.437	[19]
	Formate and acetate	Formate	β-farnesene	14.8	[20]
S. cerevisiae	Formate	Glucose	Free fatty acids	10.1	[23]
	Acetate	Glucose and formate	Free fatty acids	6.6	This study

). This indicated that the lower concentration of formate feeding led to a more efficient utilization of acetate, thereby enhancing the overall yield of FFAs. These data suggest that optimizing the formate supply can significantly impact the metabolic efficiency of the system, which may be potentially due to a reduced metabolic burden or improved metabolic channeling toward FFA synthesis. An unforeseen yet fascinating revelation emerged when we scrutinized the fatty acids’ profile. The organic phase products, especially the unsaturated fatty acid fraction, experienced varying degrees of increase (Figure S3). This alteration in fatty acid profile could have implications in varied applications, be it in biofuel efficiencies or tailored nutritional compositions. Additionally, we believe that the FFA yield in our study can be further enhanced. This could be achieved by optimizing the metabolism of acetate, such as enhancing the catalytic activity of acetyl-CoA synthetase (ACS) and introducing additional metabolic pathways that are capable of metabolizing acetate to improve substrate conversion efficiency. Meanwhile, optimizing the fatty acid production pathway could further refine the metabolic flux and energy supply, thereby providing more abundant substrates and energy for FFA production, which would, thus, increase the yield.

A noteworthy observation that accentuated the success of our optimized approach was the conspicuous absence of ethanol during this fermentation phase. Ethanol, often a byproduct in yeast metabolic pathways, can sometimes divert the metabolic flux, thus affecting the desired product generation. Its absence underscored the fact that our engineered strain was single-mindedly fixated on its task, thus showcasing an uncanny ability to transmute formate and acetate into high-energy compounds with remarkable efficiency.

In summary, these results are not just incremental improvements, but they also signify a transformative leap in our quest for sustainable bio-production. It amplifies the notion that, sometimes, the path to peak efficiency requires a blend of innovative strategy and subtle recalibration.

4. Conclusions

In the burgeoning field of biotechnology, our study represents a significant leap forward by demonstrating the adept conversion of formate and acetate into free fatty acids (FFAs), which was achieved by utilizing the microorganism Saccharomyces cerevisiae. By strategically pivoting our approach, i.e., by employing glucose as the primary carbon substrate for preliminary cellular proliferation, as well as by subsequently transitioning to the exclusive utilization of formate for energy requisites when paired with acetate as the principal carbon source, we recorded an encouraging FFA yield of 6.6 g/L. Remarkably, upon synergistically deploying both glucose and acetate as carbon scaffolds while capitalizing on formate for cellular energy needs, we witnessed an increase in FFA production, resulting in an impressive 8.0 g/L.

Our study, beyond the quantifiable metrics, offers deeper insights into the pivotal role played by formate, primarily in terms of it serving as an energy linchpin via its oxidation. Building on the foundations of our previous scholarly endeavors, we postulate that the current assimilation metrics of formate, while promising, have room for enhancement. Earlier explorations in the field have highlighted formate’s potential when used as the primary carbon substrate. Concurrently, acetate, especially given its potential to be sourced from CO₂ via innovative electrocatalytic procedures, looms large as a future-ready carbon source.

However, like any pioneering research, our study experienced some challenges. The biological nuances of Saccharomyces cerevisiae, particularly its intricate relationship with acetate, unveiled certain conundrums. During the scaled-up fermentation, an inability to sustain or escalate cellular proliferation was discerned. This limitation could potentially cap the ceilings of chemical production. In light of these findings, we advocate for the pursuit of adaptive laboratory evolution methodologies. Such avenues hold the promise of refining the microorganism’s propensity for acetate conversion, as well as serving as a potential means for enhancing its resilience against potential metabolic stresses.

Looking at future biotechnological advancements, we can identify a bifurcated objective. The initial focus should be on optimizing and augmenting the metabolic conversion matrix of acetate; simultaneously, efforts should focus on strengthening the assimilation of formate. Our overarching aspiration is to integrate the single- and double-carbon chemicals that are derived from CO₂, in which the groundwork for the advent of third-generation biorefineries will be laid. By achieving this, we envisage a future wherein biorefineries are not just experimental setups but are also pivotal pillars in the global initiative to address the twin challenges of environmental conservation and energy sustainability.