Rediscovering Acetate Metabolism: Its Potential Sources and

Review Cite This: J. Agric. Food Chem. 2018, 66, 3998−4006

pubs.acs.org/JAFC

Rediscovering Acetate Metabolism: Its Potential Sources and Utilization for Biobased Transformation into Value-Added Chemicals Hyun Gyu Lim,†,§ Ji Hoon Lee,‡,§ Myung Hyun Noh,† and Gyoo Yeol Jung*,†,‡ †

Department of Chemical Engineering and ‡School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, 77 Cheongam-Ro, Nam-Gu, Pohang, Gyeongbuk 37673, Korea ABSTRACT: One of the great advantages of microbial fermentation is the capacity to convert various carbon compounds into value-added chemicals. In this regard, there have been many efforts to engineer microorganisms to facilitate utilization of abundant carbon sources. Recently, the potential of acetate as a feedstock has been discovered; efforts have been made to produce various biochemicals from acetate based on understanding of its metabolism. In this review, we discuss the potential sources of acetate and summarized the recent progress to improve acetate utilization with microorganisms. Furthermore, we also describe representative studies that engineered microorganisms for the production of biochemicals from acetate. KEYWORDS: acetate, metabolic engineering, glyoxylate cycle, biomass hydrolysate, gas fermentation, syngas

1. INTRODUCTION Microbial processing is highly promising as it enables the production of a wide range of value-added biochemicals such as fuels, commodity chemicals, and polymers.1−4 With the remarkable advances in genetic engineering, microbial production is expected to overcome current challenges related to the depletion of fossil fuels. To realize an economically viable biological production of these chemicals, utilization of abundant and low-cost feedstock is important to consider to meet the strong demand for such chemicals. For a feedstock, several sugars (e.g., xylose, arabinose, galactose, and glycerol) which are plentiful in lignocellulosic and marine biomass or industrial byproducts have been suggested.5−9 In addition to these sugars, acetate could also be an alternative feedstock. Currently, vast amounts of acetate (12.9 million metric tons a year) are synthesized via mainly chemical routes such as methanol carbonylation, ethylene oxidation, and alkane oxidation. The United States price of acetate ($350−450 per ton) is already lower than the U.S. price of conventional sugar ($500 per ton for glucose);10−12 indeed, it is possible to obtain acetate at little or no cost from many sources.13−15 Thus, acetate could be a suitable feedstock for bioprocessing; its utilization by microorganisms should be studied for the efficient conversion of acetate into more value-added chemicals. In this review, we discuss potential sources to obtain acetate. We also give an overview of acetate metabolism in microorganisms and summarize the recent efforts to improve its utilization. Finally, we describe certain recent examples in biochemical production using acetate as a sole carbon source and as a cosubstrate with sugars.

the concentration of acetate often varies; it has been reported that typical hydrolysates contain more than 10 g/L of acetate.19 However, both acetate itself and acetate-rich hydrolysate are not properly utilized as acetate inhibits cell growth and product formation during fermentation. Thus, efficient utilization of acetate should be inevitably considered. In addition, acetate can be derived from the anaerobic digestion of biomass from wastes (e.g., animal manure, sewage sludge, and organic waste from the food and agroindustries).20,21 Anaerobic digestion is a stepwise biochemical degradation of biomass; fermentation with various microorganisms (capable of decomposing polysaccharides and producing several metabolites such as acetate and methane) can recycle the organic wastes into a renewable resource such as biogas.22 One important step in anaerobic digestion is acetogenesis; large organic compounds are digested to form acetate, which can be used for further processes. Although the acetate is currently utilized as a carbon source for methanogens to produce methane, the downstream processes could be more diversified using engineered microorganisms for its conversion into more value-added chemicals. Fermentation of industrial syngas including carbon monoxide (CO), carbon dioxide (CO2), and hydrogen (H2) is another promising source of acetate. There are several acetogenic microbes such as Acetobacterium and Clostridium which are capable of growing with syngas as the sole carbon source via the Wood−Ljungdahl pathway.23,24 This route has two branches: one is a carbonyl branch that converts CO2 into CO by reduction with a carbon monoxide dehydrogenase (CODH); the other is a methyl branch that converts CO2 into formate using the methyl-corrinoid iron−sulfur protein (methyl-CoFeS-P). From each branch, the resulting metabolites (CO and formate) are condensed to yield acetyl-CoA. Finally, acetate is

2. POTENTIAL SOURCES OF ACETATE Acetate is abundant in the hydrolysate of biomass (Figure 1); rigid polymers comprising lignocellulosic biomass can be decomposed by acid or alkali treatment to yield monomeric sugars.16 During this process, acetate is formed as a major unwanted byproduct from the deacetylation of hemicellulose.17,18 Depending on the pretreatment process for biomass, © 2018 American Chemical Society

Received: Revised: Accepted: Published: 3998

January 24, 2018 April 9, 2018 April 11, 2018 April 11, 2018 DOI: 10.1021/acs.jafc.8b00458 J. Agric. Food Chem. 2018, 66, 3998−4006

Review

Journal of Agricultural and Food Chemistry

Figure 1. Potential sources of acetate. Acetate is greatly abundant in biomass hydrolysate. In addition, various one-carbon gases could be converted to acetate via microbial fermentation or electrosynthesis.

Table 1. Microbial Production of Acetate from Various C1 Gases strain

carbon and electron source

culture condition

titer (time)

reference

A. woodii A. woodii A. woodii C. aceticum C. ljungdahlii M. thermoacetica Eubacterium limosum Sporomusa ovata microbial community from wastewater Methylomicrobium alcaliphilum 20Z Methanosarcina acetivorans

CO + formate CO2 + H2 CO2 + H2 CO + H2 CO + CO2 + H2 CO + CO2 CO CO2 + electricity CO2 + electricity CH4 + O2 + N2 CH4

anaerobic, 120 mL vial, 30 °C anaerobic, 2 L bioreactor, 30 °C, 400 rpm anaerobic, 2 L bioreactor 30 °C anaerobic, >50 mL vial, 30 °C, 200 rpm anaerobic, 2 L bioreactor, 37 °C, 500 rpm anaerobic, 1 L bioreactor, 60 °C anaerobic, >0.2 L bioreactor, 37 °C electro-bioreactor electro-bioreactor microaerobic, 250 mL vial, 28 °C, 1000 rpm anaerobic, 28 mL tube 37 °C

3.18 g/L (150 h) 44 g/L (264 h) 51 g/L (91.2 h) 2.11 g/L (48 h) 5.43 g/L (21 d) 31 g/L (70 h) 5.31 g/L (65 h) >0.53 g/L (6 days) 10.5 g/L (20 days) 504 μmol/gDCW (60 h) 0.608 g/L (120 h)

95 96 25 97 98 99 100 26 28 33 34

source to produce acetate. Methane can be specifically metabolized by methanotrophic bacteria such as Methylomonas and Methylosinus that harbor methane monooxygenase to oxidize methane into methanol;32 methanol is assimilated via either the ribulose monophosphate (RuMP) pathway or serine pathways.31 It was demonstrated that these bacteria can produce acetate in oxygen-limited conditions33 from methane assimilation. Furthermore, a recent study elucidated that methanogenesis can be reversed to assimilate methane34 even if the microorganism is not a methanotroph. Although current acetate production is much lower than conversion from CO or CO2 (Table 1), methane fermentation has huge potential considering the abundance of methane.

produced from the resulting acetyl-CoA for energy production. Because this route can transform CO or CO2 + H2 into acetate with high yields, many efforts have been made to produce acetate with these acetogens (Table 1). Recent research has successfully accumulated 51 g/L acetate by culturing recombinant Acetobacterium woodii with CO2 + H2.25 Alternatively, acetate can be produced from CO2 fixation aided by electricity through a process called microbial electrosynthesis (MES). This process often requires unique microorganisms (e.g., Sporomusa ovata) that can directly accept electrons from a graphite electrode through a biofilm.26,27 These microorganisms can produce several organic compounds, including acetate, from CO2 and electricity.26 More recently, electricity-based acetogenesis from CO2 was demonstrated by cultivation of microbial community obtained from the wastewater;28,29 Sulf urospirillum species were prevalent in the MES reactor, but the detailed mechanism is still unclear. So far, microbial electrosynthesis process has successfully demonstrated acetogenesis (10.5 g/L over 20 days).28 Because the electricity can be generated in environmentally friendly ways, acetate production from CO2 has a huge potential in the reduction of greenhouse gases. Furthermore, methane, which is highly abundant in natural and anthropogenic sources,30,31 can be utilized as a carbon

3. ACETATE UTILIZATION IN MICROORGANISMS Microorganisms consume sugars during their growth and often secrete several fermentation products for many reasons, including energy production and cofactor recycling.35 When sugars are depleted, the metabolism is shifted to reassimilate the secreted products. In the case of acetate, extracellular acetate can be metabolized by microorganisms as a form of acetyl-CoA via two routes catalyzed by acetate kinasephosphotransacetylase (AckA-Pta, encoded by ackA and pta, respectively) or acetyl-CoA synthetase (Acs, encoded by acs) 3999

DOI: 10.1021/acs.jafc.8b00458 J. Agric. Food Chem. 2018, 66, 3998−4006

Review

Journal of Agricultural and Food Chemistry (Figure 2).36 Although both pathways can convert acetate to acetyl-CoA, there are obvious differences. AckA-Pta is a

glyoxylate is condensed with additional acetyl-CoA to yield malate. Eventually, either succinate or malate can be converted into oxaloacetate, which is a precursor for another round of the cycle; the remaining molecule can be converted to pyruvate for gluconeogenesis by enzymes such as malic enzyme.50 Various cell building blocks can be produced from pyruvate via gluconeogenesis or the anaplerotic reaction. Therefore, microorganisms could grow using acetate as a sole carbon source. Additionally, it has been reported that a few bacteria utilize other cycles such as the ethylmalonyl-CoA cycle and the methylaspartate cycle, similar to the glyoxylate cycle.51,52

4. IMPROVING ACETATE UTILIZATION IN MICROORGANISMS 4.1. Redesign of the Acetate Utilization Pathway for Improved Assimilation. Although microorganisms are able to consume acetate, the utilization rate is much slower than that of sugars.41 This indicates that the utilization pathway should be redesigned for improved utilization. To harness microbial acetate assimilation, the irreversible acetate assimilation pathway (involving Acs) has mainly been up-regulated by acs overexpression.8,53−55 This strategy was generally effective to increase the assimilation rate; one study on E. coli observed, upon overexpressing acs, significantly improved acetate consumption, growth rate, and biomass accumulation in both minimal medium (M9) and rich medium (LB) supplemented with 64−124 mM (3.78−7.32 g/L) acetate compared to the wild-type strain.53 Another study also evaluated acs overexpression in S. cerevisiae,54 and similar improved effects such as shortened lag phase and increased growth rate were observed even with higher acetate concentration (140 mM, 8.27 g/L). When sugars coexist in the medium, the effect of acs overexpression can be limited due to protein-level regulation of Acs. In some microorganisms, including Salmonella enterica and E. coli, the activity of Acs is negatively controlled by protein lysine acetyltransferase (Pka, encoded by pka).56,57 Pka inactivates functional Acs by acetylation of a lysine residue56 to allow secretion of acetate. Thus, this regulation can be a bottleneck for acetate utilization. Recently, the effect of chromosomal deletion of pka was investigated in E. coli.57 The study elucidated that this deletion was highly effective in promoting reassimilation of acetate when combined with TCA cycle activation. Particularly, no growth defect was observed with this engineering.57 While the activation of the Acs pathway was beneficial to improve acetate assimilation, there was no clear result to support that acetate utilization was improved by activation of the AckA-Pta pathway. Conversely, E. coli overexpressing ackA and pta showed a severely retarded growth rate during acetate utilization.8,58 The reason for this result has not been identified. Probably, the reversible conversion of acetate to acetyl-CoA catalyzed by AckA-Pta is not a rate-limiting step; overexpression of unnecessary genes caused a metabolic burden to cells. Thus, further detailed study is required to facilitate acetate utilization with this pathway. 4.2. Activation of the Glyoxylate Cycle for Improved Acetate Assimilation. The glyoxylate cycle is not active because the transcription of the related genes (aceBAK) is negatively controlled by IclR (isocitrate lyase repressor).59,60 However, as described in Section 3, the glyoxylate cycle is a critical pathway during acetate utilization and thus should be active to improve acetate assimilation. The most common approach to activate this pathway is the removal of transcrip-

Figure 2. Overall pathway for acetate metabolism in microorganisms. Red colored enzymes are important during acetate assimilation. Symbols: CoASH, coenzyme A; Pi, phosphate; PPi, pyrophosphate.

reversible pathway for bidirectional conversion between acetate and acetyl-CoA;37 therefore, acetate can be produced or consumed through this pathway. This is a major pathway for acetate assimilation as AckA has a Vmax higher than that of Acs.38,39 Escherichia coli strains impaired in this route show growth defects when they are cultivated in acetate minimal medium.38,40,41 Conversely, Acs irreversibly converts acetate to acetyl-CoA by consuming adenosine triphosphate (ATP); this pathway is responsible only for acetate assimilation. Although some microorganisms have adenosine diphosphate (ADP)producing Acs, most microorganisms have Acs enzymes that yield adenosine monophosphate (AMP) rather than ADP.42 Thus, this reaction is much more energetically expensive than AckA-Pta. Despite the energy requirement of Acs, it has a higher affinity to acetate compared to that of AckA; in case of E. coli, the Km value of Acs is 200 μM, while AckA has a Km of 7− 10 mM.43 Because of this high affinity, Acs allows cells to grow even in low concentrations of acetate;44 acs deleted mutant E. coli was not able to consume acetate below 20 mM.38 One of the important roles of acetyl-CoA is its oxidation in the TCA cycle to produce energy and reducing cofactors.45 When acetate is the sole carbon source, the acetyl-CoA from the AckA-Pta or Acs pathway should be further transformed into the cellular building blocks with longer carbon chains (e.g., pyruvate). Some anaerobic bacteria are able to directly elongate the carbon chain of acetyl-CoA to pyruvate via the condensation of acetyl-CoA and carbon dioxide.46 However, most microorganisms are not capable of converting acetate to pyruvate directly. Thus, they utilize alternative routes for this reaction. The classical pathway is the glyoxylate cycle which is known as a bypass of the TCA cycle.47,48 This cycle is mediated by isocitrate lyase (encoded by aceA) and malate synthase (encoded by aceB) (Figure 2).49 In the cycle, isocitrate is cleaved by isocitrate lyase to glyoxylate and succinate; then, 4000


C. albidus

Y. lipolytica E. coli ΔfadE/pYX26(tesA↑)/pYX30(acs↑)

E. coli BL21(DE3)/pYJM66(QHS1↑)/ pYJM14(ERG12↑, ERG8↑, ERG19↑,IDI1↑)/ pYJM67(ACS↑) mixed culture of glycogen accumulating organisms

E. coli/ΔsdhAB/ΔmaeB/ΔiclR/gltA↑

E. coli W/ΔiclR/pCDF(cad↑)/ pCOLA(gltA↑, aceA↑)

E. coli JCL260/pAL953(ackA-pta↑)/pAL603(alsS-ilvCD↑, kivdadhA↑)/pAL991(ATF1↑) E. coli D452-2/ XYL1↑/XYL2↑/XKS1↑/ pRS425(adhE↑)

acetate

acetate acetate

acetate

acetate

acetate

acetate + glucose

acetate + xylose + glucose acetate + glucose

E. coli PB12/ΔpykAF/ΔppsA/ΔtyrR/pheAev2↑/ pJLB(aroGfbr↑, tktA↑)

Trichosporon cutaneum AS 2.571 C. curvatus C. curvatus

acetate acetate acetate

acetate

E. coli BL21(DE3)/pET-22b+(MNEI)

strain

acetate

carbon source

Table 2. Microbial Production of Biochemicals Using Acetate cultivation of engineered E. coli strain, parameter optimization (medium composition, pH, and aeration) cultivation of oleagenous yeast strain cultivation of oleagenous yeast strain cultivation of oleagenous yeast strain, parameter optimization (medium composition and pH) cultivation of oleagenous yeast strain, parameter optimization (medium composition) cultivation of oleagenous yeast with CO2-derived acetate cultivation of engineered E. coli (overexpression of acetate utilization and fatty acid biosynthesis genes) cultivation of engineered E. coli, (overexpression of acetate utilization and βcaryophyllene biosynthesis genes) cultivation of glycogen accumulating organisms (GAOs) with low-cost waste and inexpensive mixed culture biomass such as sludge cultivation of engineered E. coli (blocking both TCA cycle and gluconeogenesis, activation of glyoxylate cycle) cultivation of engineered E. coli (comparison of acetate tolerance, overexpression of acetate utilization and glyoxylate cycle genes) cultivation of engineered E. coli (simultaneous utilization of acetate and glucose to avoid carbon loss and redox imbalance) cultivation of engineered E. coli (simultaneous utilization of acetate and xylose for balanced redox state) cultivation of engineered E. coli (Avoided conversion of pyruvate to acetate to increase pool of PEP)

strategy

aromatic compound

ethanol

isobutyl acetate

itaconate

succinate

polyhydroxyalkanoate

β-caryophyllene

fatty acid fatty acid

fatty acid

fatty acid fatty acid fatty acid

monellin

product

8.08 g/L

19.7 g/L (120 h) >40 g/L (88 h)

3.57 g/L (88 h)

0.41 g/g DCW (120 h) 7.22 g/L (72 h)

18 g/L (>94h) 0.450 g/L (100 h) 1.05 g/L (72 h)

0.74 g/L (96 h)

180 mg/L (22 h) 4.4 g/L (72 h) 4.2 g/L (72 h) 10 g/L (60 h)

titer (time)

94

93

90,104

8

89

84

87

85 58

103

101 101 102

86

reference

Journal of Agricultural and Food Chemistry Review

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Journal of Agricultural and Food Chemistry tional regulation on aceBAK by deletion of iclR.61,62 E. coli with iclR deletion showed a higher growth rate and acetate consumption compared to the parental strain.8 Moreover, activation of the glyoxylate cycle via iclR deletion is also effective in the presence of sugars; therefore, this approach was used to minimize acetate formation by facilitating acetyl-CoA assimilation.63,64 In addition to IclR, there is another transcriptional regulator (FadR) which negatively controls the activity of the glyoxylate cycle in E. coli.65,66 FadR primarily regulates genes involved in fatty acid metabolism and can either activate synthesis or repress degradation. Because degradation of fatty acid, which yields the acetyl-CoA, and glyoxylate cycle are closely connected, they share the same regulation under FadR. When fadR was deleted, elevated activity of both isocitrate lyase and malate synthase were achieved.67 Furthermore, reduced acetate accumulation as well as higher biomass by fadR deletion was also observed when E. coli was cultivated in glucose medium, suggesting improved acetate (re)utilization.68,69 Although the effect of fadR deletion has not been investigated using acetate as a sole carbon source yet, this deletion could be applicable to enhance acetate assimilation in microorganisms. 4.3. Improving Acetate Tolerance to Facilitate Its Utilization. Another major drawback in acetate assimilation is its toxicity to microorganisms. Acetate inhibits cell growth,36 and this inhibition is known to vary depending on the type of microorganism.70,71 For E. coli, growth retardation is observed even with 0.5 g/L acetate.72 There are several explanations for this toxicity: (1) the neutralized form of acetate can easily penetrate the cell membrane, resulting in an unbalanced transmembrane pH potential.73 (2) Acetate induces a signal causing cells to enter the stationary phase.74 (3) Acetate inhibits production of both endogenous and heterologous proteins.36 (4) Acetate reduces the pool of intracellular amino acids such as methionine and arginine.75−77 Nevertheless, microorganisms should be viable even with the inhibition to be efficient cell factories. Therefore, there have been studies to achieve tolerance to high concentrations of acetate. The strategies can be adapted to improve acetate utilization. To overcome the toxicity of acetate to microorganisms, genome-wide studies were conducted. As growth is a key objective of microorganisms, adaptive evolution could be an effective strategy to identify beneficial mutations for acetate tolerance and rapid growth. Prior to the evolution process, 18 different E. coli strains were cultivated and their growth rate compared using a medium containing 85 mM (nearly 5 g/L) acetate as a carbon source.71 The growth rates were dramatically different among the strains; the E. coli C (ATCC8739) strain grew much faster (0.41 h−1) than the others; the lowest growth rate was 0.15 h−1, which is a 2.73-fold reduction. After this initial evaluation, the best strain was then evolved to further improve the utilization of acetate. After 450 generations of cultivation in a pseudosteady-state chemostat, the growth rate was increased to 0.51 h−1 (1.24-fold increase), suggesting emergence of potential mutations.71 To elucidate an effective mutation, whole genome sequencing of the evolved strain was conducted. From the result, it was concluded that a single amino acid change (S266P) in the α subunit of the RNA polymerase (encoded by rpoA) was responsible for the improvement; it is known to affect expression of genes of the acetate metabolism.78,79 Another genome-wide analysis was conducted in E. coli.15 Initially, they constructed a plasmid-based library which

contained randomly fragmented genomic DNA (125-nucleotide resolution) for homologous overexpression.80 Then, the cells harboring the plasmid library were cultivated in acetate medium with modest selection pressure (1.75 g/L acetate) where the growth rate was reduced by 40%. After 4 rounds of enrichment during 72 h, nearly a half of random colonies showed improved growth rate.15 Microarray-based identification of enriched inserts revealed multiple potential genes whose expression might offer acetate tolerance. For example, genes for cell wall synthesis (murC and murG), transportation (yjdL and cadA), amino acid synthesis (argA), and acetate assimilation (acs) were selected.15 In accordance with the previous observation of argA overexpression, restoring the pool of amino acids can be effective to achieve acetate tolerance; simple addition of amino acids such as methionine or glycine could recover the reduced cell growth.81 As one of major causes is known to a malfunction of methionine synthase (MetE, encoded by metE) in nonideal growth condition,82,83 Mordukhova et al. screened a mutant MetE which can maintain its activity in the presence of acetate.75 Initially, randomly mutagenized metE was constructed by error-prone PCR. Then, E. coli strains with the library were enriched under the presence of 20 mM sodium acetate. The growth selection yielded a strain with an evolved MetE (V39A, R46C, T106I, and K713E); the enzyme showed improved activity as well as stability, which can allow enhanced growth rate. Additionally, the authors observed that introduction of a mutation (C645A), identified by another group,82 showed synergistic effect to confer acetate tolerance.75

5. BIOCHEMICAL PRODUCTION WITH ACETATE UTILIZATION 5.1. Biochemical Production with Acetate As the Sole Carbon Source. As most microorganisms are already able to utilize acetate, biochemicals can be produced from acetate by replacing conventional feedstock via the native assimilation pathways; there have been several attempts in production of various biobased products from acetate as a carbon source (Table 2). For example, it was demonstrated that one type of biodegradable polymer, poly(3-hydroxyalkanoates) copolymers, was produced by mixed fermentation of glycogen accumulating bacteria using acetate.84 In addition, long-chain triacylglycerols were successfully produced by the oleaginous yeast, Yarrowia lipolytica, with acetate.85 Furthermore, protein production was also demonstrated; E. coli BL21 strain successfully produced a single chain monellin (MNEI) protein, a possible replacement for sugar in the food industry.86 Biochemical production from acetate was accelerated by engineering the acetate utilization pathway. The overexpression of acs was the fundamental approach to utilize acetate in most cases. For example, several cases of fatty acid production were demonstrated with the overexpression of acs. In E. coli, tesA (which encodes acyl-CoA thioesterase) and acs were overexpressed to produce fatty acids from acetate.58 The developed strain produced 1 g/L of fatty acids from acetate with ∼20% of the theoretical maximum yield. Further cultivation of the developed strain was also conducted to utilize acetate contained in the acid-hydrolysate of giant reed and anaerobically digested sewage sludge; the strain produced 0.43 and 0.17 g/L of fatty acids from each source. More recently, multiple acs genes from Salmonella typhimurium, Acetobacter pasteurianus, and E. coli were tested for acetate utilization.87 These genes were expressed in E. coli; acs from A. pasteurianus resulted in the 4002


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Journal of Agricultural and Food Chemistry

Moreover, a genetic modification of the metabolic pathway was reported to increase phosphoenolpyruvic acid (PEP) availability by cofermentation of glucose and acetate. Generally, the biosynthesis of aromatic compounds starts from PEP; supplementation of PEP is necessary to achieve high production. During the study, conversion of PEP to pyruvate was blocked by the deletion of the ptsHIcrr operon, pykA in E. coli, to accumulate PEP.94 With these mutations, the cell is not able to grow with glucose because it cannot produce acetylCoA from pyruvate. Instead, acetate was added into the medium to fuel the TCA cycle. Thus, the developed strain should simultaneously utilize glucose and acetate for its growth. This approach was effective to increase the PEP pool; consequently, high production of aromatic compounds was achieved (8.08 g/L) as well as high yields (0.52 C-mole of total aromatic compounds/C-mole of glucose and acetate).94

fastest growth rate among them, probably due to its high activity. Thereafter, further heterologous expression of genes encoding β-caryophyllene synthase (QHS1) from Artemisia annua, geranyl diphosphate synthase (GPPS2) from Abies grandis, and acetoacetyl-CoA synthase from Streptomyces sp. CL190 enabled the production of β-caryophyllene from acetate. The titer of β-caryophyllene reached 1.05 g/L with acetate during a 72 h fermentation.87 Overexpression of acs was also effective in the microalga Schizochytrium; improved fatty acid production was observed,88 indicating it can be broadly applicable to facilitate acetate assimilation. In addition to overexpression of the acs gene, further pathway engineering was undertaken to develop an efficient microbial cell factory. In this case, E. coli was engineered to produce succinate, one of the most important platform chemicals.89 Initially, the TCA cycle was blocked to accumulate succinate by the inactivation of the TCA cycle (ΔsdhAB). Alternatively, activity of the glyoxylate cycle was enhanced by chromosomal deletion of iclR. Further engineered strains such as those with ΔmaeB (inhibiting gluconeogenesis), ΔpoxB (preventing acetate reproduction), and overexpression of gltA (accelerating acetyl-CoA commitment into TCA cycle) were created to increase carbon flux toward succinate production. Through these attempts, the resultant strain was able to produce 7.3 g/L succinate from 20 g/L of initial acetate. 5.2. Biochemical Production with Acetate as a Cosubstrate. During sugar fermentation, acetate can be additionally supplemented as a cosubstrate to produce biochemicals. Specifically, acetate utilization is highly promising to increase the theoretical maximum carbon yield during the production of biochemicals whose main precursor is acetylCoA. When acetyl-CoA is produced from pyruvate, there is an inevitable carbon loss in the form of carbon dioxide. Conversely, there is no carbon loss during acetyl-CoA production from acetate. Thus, mole-based theoretical maximum yield with acetate utilization is 33% higher than for sugar utilization for the production of acetyl-CoA. This strategy was designed and applied to produce isobutyl acetate (IBA).90 For IBA production, acetyl-CoA should be condensed with isobutanol; acetate was intentionally added to supply acetylCoA rather than coming from pyruvate. For this, three possible acetate utilization pathways (Acs, AckA-Pta, and AldB-MhpF) were initially employed for cofermentation with glucose. Unfortunately, overexpression of ackA-pta solely enabled simultaneous utilization.37 Nevertheless, this strategy effectively increased the IBA production; the cofermentation showed a higher titer (13.9 g/L IBA) and carbon yield (47.8%) compared to the same (11.4 g/L IBA and 35.3%, respectively) from the fermentation with glucose as a sole carbon source.90 In addition, coutilization of acetate has been applied to achieve a balanced redox state. During anaerobic fermentation, balancing the redox state is highly important for both cellular growth and biochemical production.91,92 Acetate is a more oxidized substrate than sugars and it does not produce reducing cofactors when it is converted into acetyl-CoA. This property has been adapted to minimize the surplus of NADH for anaerobic ethanol production in S. cerevisiae.93 In their study, acetate was utilized to consume surplus NADH, a major drawback in ethanol production. The introduction of the acetate reduction pathway (AdhE from E. coli, which converts acetyl-CoA into ethanol) enabled simultaneous utilization of xylose and acetate and, notably, ethanol production was also significantly improved (a 1.17-fold increase).

6. FINAL REMARKS AND OUTLOOK Acetate has been regarded as a nuisance during sugar fermentation as its accumulation leads to carbon wastage and reduced cellular performance. Therefore, its metabolism has been studied to minimize accumulation by blocking synthesis or enhancing reassimilation. Notably, the developed strategies can be successfully adapted to utilize acetate, which is supplemented from various resources. As discussed, diverse biochemicals have been produced by the utilization of native or engineered microorganisms; these efforts support that acetate can be an effective carbon source for industrial chemical production. In particular, acetate utilization can be combined with sugar utilization. This cofermentation strategy could offer many advantages in efficient utilization of lignocellulosichydrolysate, higher carbon yield, and balanced redox state. However, currently, this strategy is still in an early stage for industrial-scale production. Therefore, further studies regarding acetate supplementation and its utilization by microorganisms are required.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +82 54-279-2391; Fax: +82 54-279-5528; E-mail: [email protected]. ORCID

Gyoo Yeol Jung: 0000-0002-9742-3207 Author Contributions §

H.G.L. and J.H.L contributed equally to this work.

Funding

This research was supported by the National Research Foundation of Korea (Grant NRF-2018M3D3A1A01055754) and Global Research Laboratory Program (Grant NRF2016K1A1A2912829) funded by the Ministry of Science and ICT. Notes

The authors declare no competing financial interest.

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REFERENCES

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