Carbon efficiency is one of the main factors defining the viability of a pathway process and is the main determinant of the rate of product per unit of substrate. Two factors determine carbon efficiency: the electron balance from the substrate to the product, which can be calculated from the degree of reduction of the substrate and the product, and the fact that existing metabolic pathways are primarily designed for higher reaction rates rather than high carbon yields. The first factor is closely related to the chemical composition of the substrate and product. The second factor can be overcome by redesigning the metabolic pathway, which allows the carbon of the substrate to be retained or, in some cases, assimilated during product formation.
1. Redox balance in yeast metabolism The efficiency of metabolic pathways required for efficient biological production of chemicals depends on a variety of factors such as redox balance, energy balance, thermodynamic feasibility, stoichiometric equilibrium, flux coupling, feedback inhibition, product toxicity, kinetics, and so on. Cellular metabolism maintains cell growth and redox balance by transferring electrons from substrates to different metabolites. Therefore, the optimal biosynthetic pathway for the production of a desired metabolite should be redox-neutral and the pathway yield (YP) should be at, or very close to, the maximum theoretical yield (YE) of the substrate-target product combination.The YP is dependent on the pathway involved and is determined on the basis of its stoichiometry, whereas the YE is the maximum amount of product that can be formed from the substrate and can be calculated from the substrate-to-product ratio γS/γP calculated where γS and γP are the degree of reduction of the substrate and product, respectively. The degree of reduction can be defined as the number of available electron equivalents per carbon atom of the compound. Therefore, YE needs to take into account the electron balance for the conversion of a substrate to a product, which may require decarboxylation leading to carbon loss, or carboxylation to provide additional carbon uptake. The following figure depicts the yeast centre metabolic pathway. Figure 1. yeast central carbon metabolic pathway, highlighting the relationship between carboxylation/decarboxylation steps and changes in the degree of substrate and product reduction. The degree of reduction of the corresponding substrates, intermediate metabolites and products is indicated by the colour change from red (γ=0) to yellow (γ=4) to blue (γ=6)
According to the reduction degree of substrate and target product, it can be divided into three cases: when the substrate and target product have the same reduction degree, there is an ideal situation where the substrate is completely converted into product. That is, the actual product yield can be close to the maximum theoretical yield (YE), but the metabolic process will produce by-products for the formation of biomass and maintenance of cell growth, which will reduce the product yield. An example is lactic acid (γ = 4.0), which has the same degree of reduction as glucose (γ = 4.0). Therefore, the production process of lactate is a redox-neutral pathway with a balanced stoichiometry, while allowing for the generation of ATP, which results in a rate close to the maximum theoretical yield. Overall, for other substrate-products, it is rare to find such pathways that do not generate excessive reducing power.
When the product is more reduced than the substrate, the oxidation reactions required to form the product generate additional oxidative equivalents (NAD+, NADP+, FADH+). To reduce these oxidative equivalents, the cell needs to oxidise carbon to CO2 and/or other by-products (e.g., in the pentose phosphate pathway (PPP), the TCA cycle, or the xylulose phosphate (XuMP) cycle) to maintain redox homeostasis. This process may affect the overall efficiency of substrate conversion to target products. Examples include fatty acids, ethanol, and glycerol.
The use of glucose as a substrate to generate fatty acids, such as palmitic acid (γ = 5.75), reduces fatty acid yields due to high NADPH requirements and the release of CO2 during carbon chain extension leading to substrate loss.Yu et al [1] succeeded in increasing fatty acid yields in Saccharomyces cerevisiae up to 40% by constructing an anabolic reductive metabolic pathway characterised by a repetitive decarboxylation cycle to supply the cell with additional NADH, NADPH and ATP.
The production of ethanol from glucose also oxidises some substrates to CO2 and glycerol due to the need to supply NADH. However, the natural yeast pathway for fermenting ethanol retains the degree of reduction of glucose (γ = 4.0), with an overall average reduction of γ = 4.0 when CO2 and ethanol are the final products.Thus, the metabolic pathway is very efficient from a yield perspective, converting only 4-5% of the carbon source to glycerol. Similarly, when 1,2-propanediol (1,2-PDO) (γ=5.33) was produced by brewer’s yeast using glycerol (γ=4.66) as the sole carbon source, the metabolic engineering modification provided additional NADH to facilitate 1,2-PDO synthesis, achieving the highest yields to date in yeast of >4 g/L 1,2-PDO.
When the product is reduced below the substrate, both reducing equivalents and product are generated during the production of the product. A common mechanism for re-oxidising excess reducing equivalents is through oxidation by the respiratory chain, generating excess ATP and/or releasing heat. As a result, the product yield is below the theoretical maximum achievable with available electrons. Alternatively, the excess reducing equivalents can be consumed by reducing part of the carbon source to reducing by-products. This substrate-product combination has the potential to fix carbon to increase the yield of the target metabolite. As in the production of citric acid (γ = 3.0) from glucose, the energy spillover due to the formation of NADH means that the cell can simply gain energy by making the target compound at the cost of yield loss. The poor efficiency of the natural biochemical pathway for the synthesis of citric acid therefore represents an opportunity to achieve close to the maximum theoretical rate of gain that can be achieved by fixing carbon.
Therefore, substrates for use in the desired product can be selected based on γS and γP maximising yield. The preferred substrate of yeast, glucose, can be used to synthesise products with the same γ as glucose such as ethanol (plus CO2) or lactic acid. Although glucose is the preferred substrate, glucose competes directly with food or feed production. Therefore, several cheaper carbon sources such as glycerol, methanol and CO2 are considered promising substrates.
Methanol (γ = 6.0) is a C1 feedstock with a high degree of reduction. One of the main advantages of using methanol as a carbon source is its reducing power, which in microorganisms such as methylotrophic yeast forms NADH and generates ATP.However, since the first reaction of the pathway is the oxidation of methanol to formaldehyde using oxygen as an electron acceptor, yeast loses one NADH for each serving of methanol taken up.Recent studies have shown that Komagataella phaffii was able to utilise methanol more efficiently by overexpressing endogenous methanol dehydrogenase (Adh2) in an alcohol oxidase-deficient strain (Mut-), which resulted in the production of additional NADH and ATP per portion of methanol.This allowed the Mut-Adh2 strain to increase the intensity of production of heterologous proteins under conditions of low oxygen consumption and heat emission.
Another promising carbon source is CO2, which is a highly oxidised compound (γ=0) that can be reduced by autotrophs to produce organic compounds for biosynthesis. Therefore, one way to introduce CO2 into yeast metabolism is co-substrate conversion, which converts CO2 along with another carbon source into a product with a lower degree of reduction than the co-substrate. In the biosynthesis of organic acids, which have a lower γ than glucose, such as citric, maleic and succinic acids, this strategy allows for the incorporation of CO2 into industrial fermentation processes to increase carbon yield.
2. How to balance the degree of product reduction? The evolution of metabolic processes in microorganisms is usually based on the rapid growth of cells rather than the production of specific products. Therefore, the cell favours rapid metabolism over high carbon yield. Therefore, the ability of cells to improve carbon retention during metabolism is one of the biggest challenges in metabolic engineering, which prevents microbial factories from achieving high-yield chemical production. This paper discusses the metabolic engineering of yeast aimed at maximising carbon retention, including CO2 fixation as well as avoiding non-essential decarboxylation steps in the cell.2.1 Integration of inorganic carbon into the cellular metabolism using CO2 as a substrate There are different pathways: a CO2 molecule forms organic compounds by carboxylation; CO2 is converted by reduction to formic acid or CO, which can be assimilated into biomass. Carboxylation reactions are catalysed by carboxylases. such as RuBisCO in the CBB cycle of the autotrophic CO2 fixation pathway or the pathway enzymes Pck and Pyc, which are involved in the provision of central metabolic precursors.The carbon reduction principle is that CO2 is reduced to formic acid or CO by formate dehydrogenase or CO dehydrogenase, as in the reduced acetyl coenzyme A pathway.2.1.1 Expression of Heterologous CBBase Enzymes for CO2 Fixation in YeastEthanol Production in S. cerevisiae In S. cerevisiae ethanol production, Guadalupe-Medina et al [2] utilised CO2 as an electron acceptor to harness excess reducing power, i.e., the conversion of CO2 to the PPP pathway intermediate metabolite Ru5P by the CBB cycle pathway enzymes RuBisCO and Prk, resulting in a 10% increase in ethanol production and a 90% decrease in the production of glycerol as a by-product.Xia et al [3 ] found a redox imbalance during anaerobic fermentation when xylose was used as substrate for ethanol production. Expression of RrRuBisCO and SoPRK enabled the reuse of CO2 from pyruvate decarboxylation and reduced the yield of the by-products xylitol and glycerol.Gassler et al[4] constructed a functional CBB cycle in the methylotrophic yeast K. phaffii, which provides energy and reducing power via methanol and produces lactic acid and malonate using CO2 as a carbon source.2.1.2 Reduction Glycine pathway
The reductive glycine pathway is considered to be the most efficient pathway for aerobic growth using formic acid.All the enzymes of the pro-glycine pathway are present in S. cerevisiae, but it cannot use formic acid as a substrate for growth. Overexpression of the endogenous pathway enzymes resulted in functional expression of the reduced glycine pathway, which allows the synthesis of glycine from formic acid and CO2 as a co-substrate to sustain the growth of glycine-deficient strains. The pathway is dependent on high concentrations of CO2 (10%). Recently, a naturally oxygen-resistant reduced glycine pathway was identified in K. phaffii, however the natural activity of this pathway is not sufficient to support cell growth.
2.1.3 Reduced branch of the TCA cycle (rTCA)
The reduced TCA cycle (rTCA) is a CO2 fixation pathway found in prokaryotes. rTCA is the reverse process of the oxidised TCA cycle and forms one acetyl coenzyme A molecule by fixing two CO2 molecules. So far, the complete reverse TCA cycle has not been realised in yeast. Partial rTCA was realised in Saccharomyces cerevisiae to produce succinic acid and malic acid. yan et al [5] overexpressed the genes encoding the first three enzymes of the Pyc2 and rTCA cycle, Mdh3R, EcFumC, and FrdS1, in Pdc and Fum1-deficient strains, which resulted in a yield of succinic acid up to 13 g/L with a yield of 0.21 mol/mol. malubhoy et al [ 5] synthesised 35 g/L of butanedioic acid at a yield of 0.63 mol/mol glycerol via the rTCA cycle pathway, while the process also achieved net CO2 fixation.
2.2 Avoiding unnecessary decarboxylation
Biological decarboxylation occurs primarily in catabolic pathways such as glycolysis, the PPP, and the TCA cycle, where the reaction releases CO2 and is often associated with oxidation to regenerate NADH and NADPH. decarboxylation also occurs in end-product precursor metabolite pathways, where decarboxylation reactions in the pathway all reduce the carbon yield from substrate to product. For example, acetyl coenzyme A, a metabolite produced by the decarboxylation reaction of pyruvate, results in a 33% loss of carbon in the form of CO2, which reduces the theoretical product yield of any process involving acetyl coenzyme A as a precursor. Such as the TCA cycle, fatty acid and amino acid biosynthesis. Therefore, in order to overcome the carbon loss in acetyl coenzyme A synthesis, researchers have avoided the unnecessary decarboxylation step by designing new carbon retention pathways.Hellgren et al [6] constructed a cyclic carbon conservation pathway (GATHCYC) based on the non-oxidative glycolysis pathway (NOG), which generates three molecules of acetyl coenzyme A from one molecule of fructose 6-phosphate (F6P), and the pathway does not lose carbon. The use of this pathway resulted in a 109% increase in 3-hydroxypropionic acid production. The introduction of the GATHCYC pathway into an n-butanol producing strain resulted in an increase in n-butanol production to 1.75 g/L and a 35.2% reduction in CO2 emissions.
3. Succinic acid production as an example
In addition to redox balance and carbon retention, thermodynamic feasibility and energy balance are key factors in the design of optimal metabolic pathways. Thermodynamic feasibility refers to the Gibbs free energy change (ΔrG’m) under physiologically relevant standard conditions and determines whether a metabolic pathway is feasible or not. Cellular energy should also be balanced to produce more of the target compound, as energy-demanding products lead to substrate carbon loss to meet energy demands, while oxidised products lead to energy excess and possibly heat dissipation. Succinic acid (SA) is a TCA cycle intermediate metabolite. In this section the focus is on different strategies for the production of SA and the ATP stoichiometry, redox balance, CO2 fixation, thermodynamic feasibility and carbon conservation are assessed for different natural and engineered SA synthesis pathways. There are three synthetic pathways for succinic acid: the oxidative TCA (oTCA) cycle, the reduced TCA (rTCA) cycle, and the glyoxalate pathway (GS). oTCA cycle has a lower theoretical maximum yield, but production of succinic acid under aerobic conditions has the advantage of low by-products and more favourable thermodynamic metabolic attributes. gS is an alternative method for the production of succinic acid that bypasses the isocitric acid and butyryl Coenzyme A to bypass the two decarboxylation steps between isocitric acid and butanediyl coenzyme A to prevent carbon loss and provide additional NADH. rTCA fixes CO2 and is twice as efficient as the oTCA pathway. It is important to note that the yield rate (YP) is a local parameter that considers only the net stoichiometry in the pathway and does not take into account carbon loss during NAD(P)H regeneration or ATP generation. However, the maximum theoretical yield (YE) is a global parameter that considers the electron balance and therefore also considers the regeneration of NAD(P)H. Therefore, in some cases, YP may be higher than YE. rTCA cycle synthesis of SA is mainly carried out by rumen bacteria under anaerobic conditions. In contrast, for yeast, the rTCA cycle is thermodynamically unfavourable and results in an inadequate supply of cellular NADH. The following figure compares the change in Gibbs free energy of SA synthesis via the oTCA cycle or the rTCA cycle with different carbon sources. This includes glucose, glycerol, xylose via partial CBB cycling through, assimilation of formic acid or methanol via the reduced glycine pathway and assimilation of methanol via the xyloglucan phosphate pathway. Figure 2. Production of succinic acid using the oxidative or reductive branch of the TCA cycle
The ability of yeast to tolerate lower pH and thus reduce the cost of SA production during downstream processing has led to the production of SA by yeast attracting widespread attention, especially the rTCA cycle which is capable of fixing CO2. Although the synthesis of SA from glucose via glycolysis and the rTCA cycle pathway can fix 1 mol CO2/mol SA, the pathway is not redox-balanced and requires an additional 1 mol NADH for every 1 mol of SA produced.An attractive alternative is to utilise glycerol as a carbon source, which can fix 1 mol CO2/mol SA via the rTCA pathway, allowing for oxidatively reduction-balanced SA production. The total reduction γ = 3.5 for the combination of glycerol + CO2 is the same as that of SA. Malubhoy et al [5] achieved a yield of 0.6 g/g glycerol by fixing CO2, which was 47.1% of the theoretical maximum.
Another way to achieve redox equilibrium is to utilise glucose and CO2 as co-substrates. If glycolysis, GATHCYC and partial TCA cycles are utilised simultaneously redox equilibrium can be achieved theoretically, with 1 mol of SA fixing 0.5 mol of CO2. however, 1 mol of SA requires the consumption of 0.33 mol of ATP at the cost of regenerated ATP, e.g. by respiration of part of the glucose. therefore, this scenario needs to be carried out under at least slightly aerobic conditions, which adds another another cost factor of the process.
Fig. 3 Redox-neutral production of butanedioic acid through a combination of glycolysis, GATHCYC, partial TCA cycle, and the glyoxylate pathway Table 1 Comparison of natural and engineered pathways for yeast synthesis of SA
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