Engineering propionyl-CoA pools for de novo biosynthesis of odd-chain fatty acids in microbial cell factories

Abstract Odd-chain fatty acids (OcFAs) and their derivatives have attracted great interest due to their wide applications in the food, pharmaceutical and petrochemical industries. Microorganisms can naturally de novo produce fatty acids (FAs), where mainly, even-chain with acetyl-CoA instead of odd-chain with propionyl-CoA is used as the primer. Usually, the absence of the precursor propionyl-CoA is considered the main reason that limits the efficient production of OcFAs. It is thus crucial to explore/evaluate/identify promising propionyl-CoA biosynthetic pathways to achieve large-scale biosynthesis of OcFAs. This review discusses the latest advances in microbial metabolism engineering toward producing propionyl-CoA and considers future research directions and challenges toward optimized production of OcFAs.


Introduction
Fatty acids (FAs) are the basic building blocks required by lipids for cell membrane biosynthesis in all organisms [1].FAs and their derivatives can be produced from the second-generation biomass feedstocks (abundant lignocellulosic biomass) by biorefining and are regarded as promising alternatives to fossil fuels [2].Odd-chain fatty acids (OcFAs) and their derivatives are used in a variety of applications, such as the manufacture of agrochemicals, industrial chemicals, flavor and fragrance intermediates, and pharmaceuticals [3,4].In addition, it is reported that OcFAs have positive effects on decreasing the risk of type II diabetes [5], coronary heart disease [6], and multiple sclerosis [7].
However, the extensive applications of OcFAs are limited by the great proportion of even-chain fatty acids (EcFAs) produced in the microorganisms [8].In the process of EcFAs biosynthesis, acetyl-CoA acts as a starter, and malonyl-CoA serves as the building block that provides the two-carbon unit to acyl-ACP.EcFAs are elongated with a series of repeated condensations of the acyl substrate with malonyl-CoA by fatty acid synthase (FAS) (Figure 1) [9].In contrast, the starting substrate for the biosynthesis of OcFAs is propionyl-CoA (Figure 1).Propionate can be used as the substrate to produce propionyl-CoA and the subsequent OcFAs [10][11][12][13][14].For example, microbial biosynthesis of OcFAs was achieved in E. coli by introducing the exogenous enzymes acyl-ACP thioesterases (acyl-ACP TEs) and propionyl-CoA synthetase (PrpE) with the supplementation of propionate [10].Similarly, the production of OcFAs in the engineered Y. lipolytica was boosted to 0.75 g/L after the optimization of propionate feeding and metabolic flux toward propionyl-CoA [11].Currently, most propionate is produced from fossil fuel via oxo-synthesis [15].However, propionate has a toxic effect on microorganisms [16,17].Recently, Park et al. carried out de novo biosynthesis of OcFAs in Yarrowia lipolytica using the aspartate/2-ketobutyrate pathway without propionate supplementation [8] which seems to be a promising strategy to develop alternative pathways to synthesize propionyl-CoA to enable large-scale production of OcFAs.In fact, in addition to the aspartate/ 2-oxobutyrate pathway, there are many potential biosynthetic pathways for synthesizing propionyl-CoA from glucose or glycerol, such as the 1,2-propanediol pathway [18], the aspartate/2-ketobutyrate pathway [19], the citramalate/2-ketobutyrate pathway [20], the methylmalonyl-CoA pathway (Dicarboxylic acid pathway) [21] and the 3-Hydroxypropionic acid (3-HP) pathway [22] (Figure 2).This review highlights different pathways in microbial engineering leading toward optimization of propionyl-CoA pools for de novo biosynthesis of OcFAs, and evaluates these different approaches in terms of efficiency and application prospects (Figure 2, Table 1).

The propionate pathway
Propionate can react with CoA or acyl-CoA (acetyl-CoA or succinyl-CoA) to form propionyl-CoA by PrpE or propionyl-CoA transferase (PCT), respectively (Figure 2(a)).The propionate pathway with PrpE (Figure 2(a), pathway a2) is more thermodynamically favorable than the propionate pathway with PCT (Figure 2(a), pathway a1).Due to the existence of endogenous propionyl-CoA synthetase/transferase or other enzymes with similar functions, many microorganisms can produce OcFAs with propionate supplementation [23,24].Moreover, 1-Propanol can be converted to propionate by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) (Figure 2(a)), and thus can also contribute to OcFAs production.
This propionate-based pathway is widely used to produce the propionyl-CoA, which is the key precursor of OcFAs [17].To date, the highest titer is 1.87 g/L in Y. lipolytica by adding propionate to produce OcFAs [14].It has been reported that in R. opacus the optimization of 1-propanol feeding increased the OcFAs content from 30 to 85% [25].However, propionate and 1-propanol supplementation are considerably more costly than glucose [26], and high concentrations of propionate and 1-propanol may inhibit cell growth [17].One alternative strategy is to produce propionate or 1-propanol from glucose, glycerol, or other carbon sources.As shown in the following sections, many articles have reported the metabolic engineering for de novo production of propionate and 1-propanol [20,[27][28][29].An E. coli strain has been metabolically engineered to synthesize propionyl-CoA through propionate, which is generated from glucose via the citramalate/2-ketobutyrate pathway (see the following section) [26].

The methylmalonyl-CoA pathway
The methylmalonyl-CoA pathway is also known as the dicarboxylic acid pathway [28], in which most intermediate metabolites are dicarboxylic acids from the TCA cycle (Figure 2(d)).In this pathway, succinyl-CoA is continuously isomerized into (R)-methylmalonyl-CoA and (S)-methylmalonyl-CoA by methylmalonyl-CoA mutase (MUT) and methylmalonyl-CoA epimerase (MCEE), and finally to propionyl-CoA under the decarboxylation of methylmalonyl-CoA decarboxylase (MMD) or transcarboxylation of methylmalonyl-CoA carboxyltransferase (MMC).In addition, it has been reported (R)-methylmalonyl-CoA can be directly converted to propionyl-CoA by YgfG (a MMD which has been found in E. coli) [38].There are three approaches for the formation of succinyl-CoA from glucose by TCA cycle: oxaloacetate to succinyl-CoA pathway in a reverse TCA cycle (d1), TCA cycle (d3), and glyoxylate shunt (d2).In these pathways, it takes 16-19 steps to produce propionyl-CoA from glucose.The difference between them is the maximum theoretical yield from glucose and the energy consumption (d1, d2, and d3 in Table 1).
The methylmalonyl-CoA pathway using MMC (d1.1) is also called the Wood-Werkman cycle, which is the main pathway of propionate production in Propionibacteria [39].A number of experiments have been performed, including overexpression of glycerol dehydrogenase (GldA) and enzymes of the methylmalonyl-CoA pathway (MMD, MUT, MCEE, and YgfG), deletion of lactate dehydrogenase (LDH) to explore propionate production in microorganisms based on the MMC pathway (d1) [40][41][42][43].All of these strategies have laid solid foundations for the biosynthesis of OcFAs using the methylmalonyl-CoA pathway.

The 3-HP pathway
The 3-HP bicycle is a natural CO 2 fixation pathway in photosynthetic green nonsulfur bacteria [22], and propionyl-CoA is a key metabolic intermediate in this pathway.As one of the sub-pathways of the 3-HP bicycle, the 3-HP pathway (e1) has been described in several literatures as a potential pathway to produce propionyl-CoA [17,44].In this pathway, acetyl-CoA is first converted to 3-HP by acetyl-CoA carboxylase (ACC) and malonyl-CoA reductase (MCR), and then to propionyl-CoA, catalyzed by: 3-hydroxypropionyl-CoA synthase (HPCS), 3-hydroxypropionyl-CoA dehydratase (HPCD), and acryloyl-CoA reductase (ACR).It takes 2 NAD(P)H and 3 ATP in 17 steps from glucose to propionyl-CoA (e1 in Table 1).3-HP can also be converted from glycerol through the dehydration and oxidation of glycerol by glycerol dehydratase (GDH) and ALDH (e2 in Figure 2(e)).The b-alanine pathway (e3) is yet another pathway for producing 3-HP by the decarboxylation of aspartate.The key enzymes in these three pathways are: HPCS, HPCD, and ACR.

Other potential pathways
In addition, the acrylate pathway (f1), the ethylmalonyl-CoA (EMC) pathway (g1) and the citramalyl-CoA pathway (g2) can also generate propionyl-CoA from glucose.The acrylate pathway originally reported as the propionate biosynthetic pathway can be found in organisms like Clostridium propionicum and Megasphaera elsdenii [27].In this pathway, lactate is directly converted to lactoyl-CoA via PCT with propionyl-CoA to form propionate, and then lactoyl-CoA is catalyzed to form propionyl-CoA under the dehydration of lactoyl-CoA dehydratase (Lcd) and reduction of ACR (Figure 2(f)).Recently, a report on the systematic mining of acetyl-CoA: lactate CoA-transferases (ALCTs) enables the acrylate pathway to produce propionyl-CoA from lactate [49].The ALCT can transfer CoA from acetyl-CoA to lactate, so that the final product of the acrylate pathway is propionyl-CoA instead of propionate.In this pathway, glucose is, in turn, converted to pyruvate and lactate and eventually forms propionyl-CoA, and the maximum theoretical yield is 2 mol propionyl-CoA per mol glucose within 14 steps.Key enzymes in this pathway are ALCT and ACR.

Limitations and key factors of the propionyl-CoA biosynthetic pathways
The efficiency of a biosynthetic pathway is usually affected by the following factors: number of reactions, driving force (NAD(P)H and ATP), thermodynamic feasibility, and activities of key enzymes.In order to achieve high yields of propionyl-CoA in validated and predicted pathways (Figure 2), it was crucial to understand the mechanism of each pathway.To enable comparative analysis, the overall stoichiometry in each pathway was plotted based on the conversion from glucose (glycerol or another economical carbon source) to propionyl-CoA (Table 1).
Through 1,2-propanediol pathways (b2, b3), propionyl-CoA also can be produced from L-rhamnose and L-fucose, which are abundant in potential biomass feedstock seaweeds [57,58].These two pathways take fewer steps but are less thermodynamically favorable than the pathway (b1) from glucose (Figure 2(b) and Table 1).Glycerol is a by-product of biodiesel, and has been regarded as an attractive and competitive carbon source [59].In this review, we list three propionyl-CoA biosynthetic pathways from glycerol.They have fewer steps and use less ATP or NAD(P)H than the glucose pathways.Among them, the glycerol/3-HP pathway is the shortest, with 5 steps (Table 1).
Thermodynamics determine the feasibility of a pathway.Challenging thermodynamic reactions (D r G 00 > 10 kJ/mol) in propionyl-CoA pathways are catalyzed by: ADH in the 1-propanol pathway (Figure 2 MCL in the EMC pathway (g1).To overcome these thermodynamic obstacles, the following strategies can be used: (i) maintaining the highest possible ratio of substrate to product concentration, and (ii) providing a strong reducing environment in cells [60].
Considerable achievements have been reported in the microbe-based production of several key intermediates in the above-discussed pathways (Figure 2), such as 1,2-propanediol, 2-ketobutyrate, 1-propanol, propionate, and 3-HP production [20,29,47,61,62].A sufficient supply of these precursors facilitates downstream reactions for efficient production of propionyl-CoA.However, there are only a few reports about engineering propionyl-CoA pools from these intermediates, with the exception of 1-propanol and propionate [17].Therefore, the work that follows can be focused on the screening and optimization of downstream enzymes in generating propionyl-CoA, including: PduCDE and PduP in the 1,2-propanediol pathway, the PDH, PflB, and PrpE in the 2-ketobutyrate pathway, MUT, MCEE, MMD, MMC, and YgfG in the methylmalonyl-CoA pathway, HPCS, HPCD, and ACR in the 3-HP pathway, ALCT in the acrylate pathway, MCL in the EMC pathway and citramalyl-CoA pathway.

Conclusion and perspective
This review has summarized recent advances in propionyl-CoA biosynthetic pathways.These different approaches have been evaluated in terms of efficiency and application prospects.Among them, aspartate/2ketobutyrate pathway (c1) is currently the only pathway who successfully produces OcFAs through the increasing of de novo propionyl-CoA pool [8].Other propionyl-CoA biosynthetic pathways show their potential for producing propionyl-CoA to enable de novo biosynthesis of OcFAs in microbial cell factories.In addition to increasing the supply of the precursor propionyl-CoA, efforts in other aspects are necessary: (i) balancing acetyl-CoA production and malonyl-CoA consumption pathways in the engineering hosts to improve the yield of OcFAs, (ii) optimizing the carbon sources and C/N ratio of the medium to improve lipid-to-biomass ratio, (iii) screening of propionyl-CoA preferred FAS enzymes or enzymatic engineering FASs to increase the ratio of OcFAs verse EcFAs, (iv) reprogramming of FA metabolism to control chain-length of OcFAs, and (v) developing more economical biomass to produce OcFAs.Further study into these directions may lead to the development of efficient production of OcFAs.Meanwhile, studies of accumulated OcFAs on lipid composition, on cell growth and on biomass production would be valuable to see what the highest ratio of OcFAs that a cell can accept.