Review
Metabolic engineering as a tool for enhanced lactic acid production Bikram P. Upadhyaya1,2, Linda C. DeVeaux1,2,3, and Lew P. Christopher1,2 1
Center for Bioprocessing Research & Development, South Dakota School of Mines & Technology, Rapid City 57701, SD, USA Biomedical Engineering Program, South Dakota School of Mines & Technology, Rapid City 57701, SD, USA 3 Department of Chemistry & Applied Biological Sciences, South Dakota School of Mines & Technology, Rapid City 57701, SD, USA 2
Metabolic engineering is a powerful biotechnological tool that finds, among others, increased use in constructing microbial strains for higher lactic acid productivity, lower costs and reduced pollution. Engineering the metabolic pathways has concentrated on improving the lactic acid fermentation parameters, enhancing the acid tolerance of production organisms and their abilities to utilize a broad range of substrates, including fermentable biomass-derived sugars. Recent efforts have focused on metabolic engineering of lactic acid bacteria as they produce high yields and have a small genome size that facilitates their genetic manipulation. We summarize here the current trends in metabolic engineering techniques and strategies for manipulating lactic acid producing organisms developed to address and overcome major challenges in the lactic acid production process. Importance of lactic acid Lactic acid, or 2-hydroxypropanoic acid, is a naturally occurring chiral organic compound that has been used in both the food and non-food industries for almost 120 years [1]. Lactic acid is approved by the U.S. Food and Drug Administration as GRAS (generally regarded as safe), so its applications in food and other chemical industries are diverse [2]. Cosmetic industries also benefit from lactic acid, as it is used in the manufacture of hygiene and esthetic products [3]. Furthermore, lactic acid has gained tremendous demand as feedstock for the chemical synthesis of poly-lactic acid (PLA) [4]. Since PLA is biodegradable, it can be utilized as an environmentally friendly alternative to petro-chemically derived plastics in the textile, medical, and pharmaceutical industries [5]. The current market price for 88% food grade lactic acid is $1400– 1600 per metric ton (see: http://www.icis.com/chemicals/ channel-info-chemicals-a-z/), with an annual demand of 130 000–150 000 metric tons, which is estimated to reach 367 300 metric tons by 2017 (http://www.prweb.com/ releases/lactic_acid/polylactic_acid/prweb9369473.htm), and over one million tons by 2020 [6]. The rapid annual Corresponding author: Christopher, L.P. ([email protected]). Keywords: metabolic engineering; lactic acid production; lactic acid purity; acid tolerance; carbon source; fermentation parameters. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.10.005
growth of approximately 20% is expected to come from the increasing demand of PLA as a biodegradable polymer. In this review, we discuss recent progress in metabolic engineering strategies, based on both evolutionary and rational methodologies, which have been effective in improving lactic acid production and efficiency in a variety of microorganisms (Figure 1). Challenges and metabolic engineering solutions for lactic acid production Based on a PLA price of $2.2/kg and factoring in the plant size, raw material cost, and capital investment, a target manufacturing cost of lactic acid has been estimated at $0.55/kg [7]. However, in order to minimize production costs, further biotechnological advances are needed to develop high-performance lactic acid producing microorganisms that enhance the fermentation and recovery efficiency in lactic acid production. Lactic acid bacteria (LAB) naturally produce lactic acid as the primary metabolic endproduct, but the chemical and optical purity can vary, and these organisms are generally fastidious, requiring costly nutritional supplementation. Lactobacillus (Lb.) sp. and Lactococcus (L.) sp. are the predominant species used for lactic acid production, but we have recently reported a high yield lactic acid producing Enterococcus isolate that is a promising candidate for metabolic engineering, and does not have the complex nutritional requirements of other LAB [6,8]. Because of their innate acid tolerance, yeasts such as Candida sp. are being developed for lactic acid production, but also require metabolic engineering to introduce and optimize pathways. We discuss here the potential of metabolic engineering as a successful tool to improve the lactic acid fermentation process in a cost-efficient and environmentally friendly way. Emphasis is given to the four major challenges associated with lactic acid production that can be addressed by employing suitable metabolic engineering strategies: (i) purity, (ii) acid tolerance, (iii) carbon source, and (iv) industrial parameters. Challenge 1: Purity of lactic acid Optically and chemically pure lactic acid is required for PLA synthesis; optical purity is a major factor that determines the physical properties of PLA. PLA with a high crystallinity and a high melting point, suitable for the production of fibers, oriented films and liquid crystals [9], could be produced from either the optically pure Trends in Biotechnology, December 2014, Vol. 32, No. 12
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Metabolic engineering
Raonal Engineering
Evoluonary engineering
Pathway redirecon
Classical strain improvement
Increased gene expression
Shotgun mutagenesis approaches
Genome shuffling
Heterologous gene expression
Whole genome amplificaon
Mulplex automated genomic engineering TRENDS in Biotechnology
Figure 1. Metabolic engineering approaches used to address challenges in lactic acid production.
L(+)- or the D( )-lactic acid isomer, but not from a racemic mixture of the two isomers. A main advantage of microbial fermentation processes [10,11] over chemical production methods [12] is the ability to produce optically pure lactic acid. However, the optical purity of lactic acid is dependent on the microbial strain utilized, and can be further optimized. The chemical purity of lactic acid is affected by the composition of the fermentation medium, which in turn is a function of the nutritional requirements of the microbial producer. Traditionally, industrial lactic acid has been produced by fastidious LAB such as Lactobacillus, which
Lignocellulosic feedstock Glucose
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Figure 2. Schematic representation of the carbon metabolic pathways in lactic acid fermentation. Broken arrows represent multiple consecutive steps in the pathways. Unshaded area (desired route): lactic acid metabolic pathway. Shaded area (unwanted route of co-product formation): (A) ethanol/acetate metabolic pathway in LAB; (B) succinate metabolic pathway in Escherichia coli.
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utilize expensive carbon (glucose) and nitrogen (amino acids) sources together with vitamins and minerals [13]. Most LAB lack the ability to synthesize growth factors, and generally have complex nutritional requirements, which results in increased purification costs of the final product. As purification costs comprise a significant portion of the overall production costs, the purity of food grade (80–90% purity) and pharmaceutical grade (>90%) lactic acid significantly influences its price [14]. Metabolic engineering solutions to Challenge 1 The laboratory model Escherichia coli has been used in a number of applications due to its ease of growth and well established methods of molecular and genetic manipulations [15–18]. However, E. coli requires metabolic engineering to direct pyruvate toward the single fermentation product of lactic acid (Figure 2). Genes encoding enzymes catalyzing conversion of pyruvate to succinate, acetate, and ethanol have been inactivated, resulting in higher production and chemical purity of the desired lactate product (Table 1). LAB have been engineered to increase the optical and chemical purity of lactic acid. Lb. helveticus in its native state produces a racemic mixture of L(+)- and D( )-lactic acid isomers. By removing the promoter region of the gene for D( )-lactate dehydrogenase (LDH), Lb. helveticus produces only the L(+)-isomer [19]. However, the complex nutritional requirements of the LAB complicate industrial processes and increase costs. To circumvent this, Corynebacterium glutamicum, which can produce lactic acid in minimal medium, has been engineered to express the D( )LDH gene from L. bulgaricus, producing D( )-lactate with >99.9% optical purity [20].
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Table 1. Major challengesa addressed utilizing metabolic engineering tools for enhanced lactic acid production Approach Rational engineering
Major challenge addressed Purity
Acid tolerance Carbon source
Evolutionary engineering
Acid tolerance
Carbon source
Outcome(s) Pure L(+)-lactic acid
Organisms Lactobacillus (Lb.) helveticus
Refs [19]
Pure L(+)-lactic acid (>99%) Titer – 97 g/l, Yield – 90.0%, Productivity – 3.17 g/l/h (Sucrose fermentation) Pure (99.9%) D(S)-lactic acid Titer – 17.92 g/l Ethanol-free lactic acid Titer – 92 g/l, Yield – 94.0% Pure (99.3%) L(+)-lactic acid Yield – 93.0% Pure (99.9%) L(+)-lactic acid Titer – 122 g/l pH 3.0: increased survival after 2 h Cell survival at pH 3.3 Xylose to produce L(+)-lactic acid Purity – 99.5%, Titer – 62.0 g/l Yield – 97.0%, Productivity – 1.631 g/l/h (Xylose fermentation) Glycerol to produce D(S)-lactic acid Purity – 99.97%, Titer – 100.3 g/l Yield – 78.0%, Productivity – 2.78 g/l/h (Glycerol fermentation) Glycerol to produce L(+) lactic acid Purity – 99.9%, Titer – 50 g/l Yield – 93.0%, Productivity – 1.3 g/l/h (Glycerol fermentation) Xylose to produce L(+)-lactic acid Purity – 99.6%, Titer – 50.1 g/l, Yield – 94.6% (Xylose fermentation) Corn starch to produce D(S)-lactic acid Purity – 99.6%, Titer – 73.2 g/l Yield – 0.85 g per g of consumed sugar Productivity – 3.86 g/l/h (Starch fermentation) Mixture of glucose and xylose to produce D(S)-lactic acid Purity – 99.5%, Titer – 74.2 g/l, Yield – 0.78 g per g of consumed sugar, Productivity – 5.6 g/l/h (25 g/l xylose + 75 g/l glucose fermentation) CO2 to produce D(S)-lactic acid Titer – 1.14 g/l (photoautotrophic condition) Xylose to produce L(+)-lactic acid Purity – 99.9%, Titer – 93.9 g/l, Yield – 91.0%, Productivity – 2.18 g/l/h (Xylose fermentation) Xylan to produce lactic acid Titer – 1.7 g/l (Xylan fermentation) At pH 4: titer increased 3-fold over wild type Cell survival at pH 4.3 Titer – Increased by 13.6% Improved cell activity at pH 3.8 Titer – 3.36 g/l, Yield – 95%, Productivity 2.25 g/l/ h At pH 4: 64.5% improvement in lactic acid titer over the wild type Titer – 12 g/l At pH 3.8: 2.6-fold increase in cell growth; Titer - 3.1-fold increase, Productivity – 5.770.05 g/l/h Xylose to produce L(+)-lactic acid Purity – 99.5%, Titer – 62.0 g/l, Yield – 97.0%, Productivity – 1.631 g/l/h (Xylose fermentation) Sucrose to produce D(S)-lactic acid Titer – 110 g/l, Yield – 73.0% Sugarcane molasses and corn steep liquor to produce L(+)-lactic acid
Escherichia coli
[18]
Corynebacterium glutamicum
[20]
Candida (C.) sonorensis
[27]
Thermoanaerobacterium aotearoense Saccharomyces cerevisiae Lactococcus (L.) lactis Lb. casei E. coli
[40] [28] [26] [48] [49]
E. coli
[17]
E. coli
[16]
L. lactis
[39]
Lb. plantarum
[50]
Lb. plantarum
[51]
Synechocystis sp.
[42]
C. utilis
[52]
Lb. brevis
[53]
Lactobacillus Lb. casei
[23] [54]
Lb. pentosus
[32]
Lb. plantarum
[55]
Lb. rhamnosus
[31]
E. coli
[49]
Lb. lactis
[24]
E. coli
[18]
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Table 1 (Continued ) Approach
Major challenge addressed
Parameters
Outcome(s) Purity > 99.0%, Titer – 75 g/l, Yield – 85.0%, Productivity – 1.18 g/l/h Cellobiose and cellulose to produce D(S)-lactic acid Titer – 80 g/l (cellobiose), 73 g/l (cellulose) Productivity – 1.66 g/l/h (cellobiose), 1.52 g/l/h (cellulose) Sucrose to produce lactic acid Titer – 135 g/l, Yield – 90.0% Starch to produce lactic acid Titer – 54 g/l Starch to produce L(+)-lactic acid Purity – 99.05%, Titer – 79.4 g/l Reduced substrate (glucose) inhibition Purity – 99.0%, Titer – 184 g/l, Yield > 90.0%
Organisms
Refs
Lb. lactis
[3]
Lb. delbrueckii
[56]
Lb. delbrueckii
[57]
Rhizopus oryzae
[58]
Lb. rhamnosus
[47]
a
Information on the main challenge addressed is provided in bold; additional outcome(s) are presented, where available.
Challenge 2: Acid tolerance of lactic acid producing organisms Glucose fermentation by homofermentative LAB requires slightly acidic to neutral pH, as low pH has an inhibitory effect on cellular metabolism, and thus lactic acid production. The majority of LAB cannot grow below pH 4, although the pKa of lactic acid is 3.78. In order to maintain a neutral pH for cell survival, lime is routinely added to the fermentors, which results in more than 90% of the lactic acid present as calcium lactate. Following fermentation, the calcium lactate-containing broth is acidified with sulfuric acid to produce lactic acid and insoluble calcium sulfate [21]. The disadvantages of this process include the production of a large amount of calcium sulfate (gypsum) as a by-product and a high sulfuric acid consumption causing corrosion problems [22]. The amount of gypsum is usually higher than the amount of lactic acid produced, and more than a ton of gypsum is generated for every ton of lactic acid, presenting a waste disposal problem and a major cost factor in the product recovery stage of commercial operations. Ideally, microbial fermentation would occur in medium with a pH at or below the pKa of lactic acid, allowing direct purification of the acid form. Metabolic engineering solutions to Challenge 2 Classical mutagenesis (Box 1) has been applied to select for variants of Lactobacillus sp. with increased tolerance to the acidified medium produced during fermentation. After UV and nitrosoguanidine treatment, these improved strains are capable of lactic acid production at rates and yields similar to those of the traditional, neutral-pH lactic acid processes [23,24]. Acidic conditions represent an environmental stress that activates specific and global stress responses, involving induction of the synthesis of several proteins [25]. In order to increase resistance to the acidic conditions resulting from lactic acid fermentation, enzymes such as trehalose 6-phosphate phosphatase from Propionibacterium freudenreichii [25] has been expressed in L. lactic resulting in 5- to 10-fold higher survivability at pH 3.0. In a similar study, the histidine decarboxylation pathway from Streptococcus thermophilus [26] was expressed in L. lactis, resulting in survival at pH levels as low as 3 in which the host cells were rapidly dying. 640
To capitalize on the natural acid resistance of yeasts, lactic acid productivity has been improved by introducing the gene encoding L(+)-LDH from heterologous sources. The bovine gene encoding LDH has been successfully expressed in both Candida (C.) utilis and Saccharomyces cerevisiae, and the gene encoding LDH from Lb. helveticus has been expressed in Candida sonorensis [27–29]. Genome shuffling (Box 1) has been a successful technique to enhance complex phenotypes while reducing the deleterious effects of accumulating extraneous mutations [23,30]. In Lactobacillus, this technique was used to lower the optimal pH of lactic acid production, without loss of yield. One population was adapted gradually to fermentation in medium with incrementally lower pH, while a second population was mutagenized with nitrosoguanidine, then selected on medium with lowered pH. Following protoplast formation, the two populations were fused. Fused isolates were selected on low pH medium and screened for high lactic acid productivity [23,31]. This resulted in strains that produced three times the wild type levels of lactic acid at pH 3.8, close to the pKa of lactic acid. Recently, whole genome amplification (Box 1), a new method for introducing random mutations using PCR and electroporation, has been used to improve the acid tolerance of Lb. pentosus, producing a strain with 95% lactic acid yield at pH 3.8, in which the wild type strain did not grow [32]. Since this technique does not rely on protoplast fusion, it could have a wider application to other species beyond the Lactobacilli. Challenge 3: Carbon source for lactic acid production As polymer producers and other industrial users usually require large quantities of lactic acid at a relatively low cost, inexpensive raw materials are essential for the economic feasibility of the microbial process. The use of starch and refined sugars (glucose) as feedstock increase the cost of lactic acid, as in a typical lactic acid fermentation process the raw material cost constitutes between 40% and 70% of the total production cost [33]. The raw materials for industrial lactic acid production need to have several characteristics, such as low cost, low levels of contaminants, rapid fermentation rate, high lactic acid yields, little or no by-product formation, and year-round availability. Since glucose or any other food-based feedstock directly compete
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Box 1. Evolutionary engineering Evolutionary engineering resembles Darwinian evolution, but with much shorter time, and thus faster progression toward a desired characteristic [59]. A very small subset of the population will have been changed, and it is unnecessary to understand the molecular or genetic mechanism behind the improved characteristics. Because of this, however, it is usually not possible to transfer specific genetic information to other strains [60]. Classical strain improvement. Selection for beneficial mutations through classical strain improvement may rely on spontaneous mutations that occur in any growing population. Alternatively, a mutagen may be applied to increase the frequency of mutations. In either case, the resulting mixed population is subjected to conditions that will select or enrich for those individual cells that have acquired the ability to survive (Figure I). Shotgun mutagenesis approaches. Unlike classical strain improvement, shotgun mutagenesis approaches can introduce many more mutations with a higher throughput, reducing the mutational load on any individual. This shortens the time to carry out the process which, along with higher sensitivity for detection, allows more mutagenized individuals to be assessed in a short time. The methods described below do not represent an exhaustive list, but provide examples of these next-generation approaches (see Figure 1 in main text). Genome shuffling. Genome shuffling utilizes recursive protoplast fusion to allow recombination between genomes so that only the desired mutation(s) may be selected [23]. Briefly, two populations are generated. One population is adapted by selecting for spontaneous mutations and the second population is generated through chemical or physical mutagenesis, creating a library of strains, presumably with many mutations per chromosome. The genomes of the two populations are then ‘‘shuffled’’ by fusing cells from each population, followed by selection for the desired trait (Figure II). Whole genome amplification (WGA). In WGA, short, random oligonucleotide primers are used to prime PCR with the genome of
(A)
X
the organism as the template. By using a non-proofreading polymerase, fragments are generated with random nucleotide changes introduced. These fragments are then re-introduced into the organism to be modified using electroporation, with subsequent selection or high-throughput screening for the desired traits [32,61]. Multiplex Automated Genome Engineering (MAGE). MAGE has automated the introduction of random, mutated genomic pieces, speeding up directed evolution. Using multiplex DNA synthesis, random mutated pieces are generated and introduced into cells, and, as with other approaches, useful mutants are selected with suitable media or in various environmental conditions.
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Figure II. Schematic representation of heterologous gene expression. Metabolic pathway system of the starting strain converts substrate (X) to intermediate product (I) but lacks the enzymatic pathway to the target product (Z). Expression of the cloned gene from an unrelated strain (capable of converting I to Z) allows the conversion of X to Z in the engineered strain.
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Strain engineered through increased gene expression TRENDS in Biotechnology
Figure I. Schematic representation of pathway re-direction and increased gene expression (A) Metabolic pathway system of the starting strain converting substrate X to co-product Y and target product Z. (B) Elimination of the enzyme pathways that convert X to Y resulting in increased levels of target product Z. (C) Overexpression of the enzymes responsible for converting X to Z resulting in increased levels of target product Z.
with the food supply chain, there is a growing interest in the use of lignocellulosic biomass (Figure 2) as a renewable and low-cost substrate for the microbial production of lactic acid [34–37]. However, the release of fermentable sugars from the complex lignocellulose polymer requires additional processing steps (e.g., biomass pre-treatment, hydrolysis and possible detoxification of lignocellulosic hydrolyzates), which increase the production cost of lactic acid. Metabolic engineering solutions to Challenge 3 A natural isolate of E. coli was engineered to remove the repressor of genes encoding enzymes required for sucrose utilization, which may allow lactate production from more inexpensive sugar sources [38]. Similarly in L. lactis, optically
pure L(+)-lactic acid was made from xylose, by elimination of the phosphoketolase pathway of xylose utilization [39]. With improved molecular biology techniques, engineering similar deletion mutations in less well-studied organisms has been effective. Thermoanaerobacterium aotearoense, a thermophile and obligate anaerobe that can convert lignocellulose directly to L(+)-lactate, was made homo-fermentative by blocking the competing formation of acetic acid [40]. Microalgae are being considered for lactic acid production, as, among other reasons, the utilization of light energy to fix carbon could potentially eliminate feedstock costs. Synechocystis sp. have been engineered (Table 1) to express either an L(+)-LDH gene to produce L(+)lactate [41] or a mutated glycerol dehydrogenase gene 641
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Box 2. Rational metabolic engineering A rational approach to metabolic engineering refers to a directed, and usually very specific, alteration of a known pathway or protein, which is expected to have a particular outcome. As such, it requires extensive genetic and metabolic information about the target organism. Usually these directed changes yield the predicted result. However, rational engineering perturbs normal metabolism and has also activated novel, sometimes cryptic pathways, revealing previously unrecognized complex cellular regulatory networks [60]. In addition, when strong selective pressures are applied, compensatory mutations present in the population may alter other existing pathways, for example, by changing substrate specificities of related enzymes [43]. Pathway re-direction. Generally, pathway re-direction involves genetic modification to eliminate competing pathways for pyruvate utilization or production, or pathways leading to accumulation of unwanted metabolites (Figure I). In addition to requiring knowledge of the existing pathways, the organism must be amenable to such manipulations. Increasingly, pathway re-direction is combined with other molecular strain improvements to optimize flux through the desired pathway. It has also been a successful approach to improve the optical purity of lactic acid. For example, Escherichia coli contains genes for production of both the D( )- and L(+)-isomers. The optical purity was increased by elimination of the pathways for the alternate isomer, or by preventing undesired utilization of the desired isomer [16]. Increased gene expression. A complementary approach to pathway re-direction is to increase the transcription of the gene(s) encoding the enzyme(s) in the desired pathway (Figure I). Increases in gene expression are most easily accomplished by cloning the gene(s) of interest, and expressing on a multicopy, extra-chromosomal element within the cell, or by replacing a native promoter with a more efficient promoter on the chromosome. Heterologous gene expression. Organisms with desirable properties for lactic acid production, but lacking key enzymatic processes, can be engineered to express non-native genes, introducing novel metabolic characteristics that improve their industrial importance (Figure II). While advances in molecular biology techniques have broadened the application of such heterologous protein expression, there is no single system that works across the board.
Adaptaon
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Starng strain
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+
+
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++
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Figure I. Schematic representation of classical strain improvement. Arrows represent selection of the desired strains. Broken squares refer to selected strains for the next round of mutagenesis. Ovals represent individuals within the population. Plus (+) represents improved characteristics; minus (–) represents undesirable characteristics.
Protoplasts fuse
Chromosomes recombine
Engineered strains
Selecon
TRENDS in Biotechnology
Figure II. Schematic representation of genome shuffling as a metabolic engineering tool for strain improvement.
to produce D( )-lactate [42], both originating from Bacillus sp. [43].
repression [44]. These parameters depend on the type of microbial producer and its metabolic pathways (Figure 2).
Challenge 4: Parameters for industrial lactic acid production The key economic drivers for a cost-efficient lactic acid production are: lactate yield (% of theoretical maximum from carbon source), volumetric productivity (g/l/h), and titer (g/l). The commercial production process requires the use of non-fastidious and robust microorganisms that can meet the industrial needs for high yield, productivity and titer of lactic acid of at least 80%, 2.5 g/l/h and 100 g/l, respectively [8]. Other important fermentation parameters include specific growth rate of the production organism, metabolite formation, ability to ferment both pentose and hexose sugars, no substrate or end-product inhibition, resistance to inhibitory compounds formed during pre-treatment of lignocellulosic biomass, and minimizing carbon catabolite
Metabolic engineering solutions to Challenge 4 As naturally high lactic acid producers, LAB such as Lactococci and Lactobacilli have been engineered to heterologously express other enzymes that improve productivity. L. lactis expressing part of the gene encoding phosphofructokinase from Aspergillus niger showed increased flux through the glycolytic pathway, resulting in increased lactate production [45]. Within the species, L. lactis IL with a disrupted phosphoketolase pathway, but enhanced pentose phosphate pathway due to expression of the transketolase genes from L. lactis IO-1, produced L(+)-lactic acid from xylose at levels close to the theoretical value [39]. Recently, Lb. helveticus was engineered to express only L(+)-lactate by deletion of the gene for D( )-LDH, and an additional copy of the gene for
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Review L(+)-LDH was inserted in its place, resulting in almost twice the total native production, and all optically pure L(+)-lactate [19]. In another study, D( )-LDH has been overexpressed in E. coli, which resulted in increased production and yield of D( )-lactate from glycerol [17]. Eukaryotic lactic acid producers, such as Candida sp., have also been engineered for the production of L(+)-lactic acid. The gene encoding the pyruvate decarboxylase of C. utilis was disrupted, which, together with heterologous expression of LDH made L(+)-lactic acid with high efficiency [29]. Genome shuffling has been used to increase the tolerance to high substrate concentrations, which is normally inhibitory to cell growth and lactic acid production [46]. Simultaneous selection for glucose tolerance and high lactic acid production yielded a strain of Lb. rhamnosus that made 70% more lactic acid than the wild type at 150 g/l glucose [47]. Critical analysis of metabolic engineering advances Acid stress and end-product inhibition are still among the major bottlenecks in commercial lactic acid production; hence, further progress in metabolic modeling and systems biology would help engineer advanced biosynthetic pathways for an improved biotechnological process. Furthermore, the successful identification of genes and proteins associated with stress response and tolerance would facilitate the design of rational approaches for metabolic engineering (Box 2). In addition, the acid tolerance and carbon substrate range of producing organisms can be broadened, which allows the design of a sustainable and environmentally sound production process. More focus should be given to the heterologous expression of enzyme and metabolic pathways in yeasts and fungi as they are more acid tolerant than LAB and naturally produce more optically pure lactic acid. As the raw material is a major cost factor in lactic acid production, future developments in metabolic engineering are expected to enhance the utilization of lignocellulosic biomass (e.g., biomass-derived hexose and pentose sugars) and biomass-derived waste streams (e.g., carbon dioxide, paper sludge, agriwaste, and food waste). In the case of lignocellulosic biomass, efforts should be made to increase microbial resistance toward inhibitory compounds formed during biomass pre-treatment and pentose fermentation, and toward carbon catabolite repression caused by heterologous sugar substrates present in biomass. However, waste streams derived from biomass would likely be the preferred feedstock for lactic acid production as (i) it eliminates the need for biomass pre-treatment and formation of unwanted inhibitory products utilizing a low-cost feedstock thereby minimizing total cost; and (ii) it utilizes waste by-product thereby alleviating pollution and greenhouse gas emission problems. From this perspective, engineering microalgae and cyanobacteria to increase their photosynthetic efficiency of lactic acid production from carbon dioxide could significantly lower production costs. Concluding remarks and future perspectives The rapidly growing demand for lactic acid dictates the need for use of robust and highly efficient microorganisms that meet the strict industrial requirements for cost-efficient lactic acid production. To address this challenge, metabolic engineering is increasingly employed by biotechnologists
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as a powerful tool that can manipulate the traits of lactic acid microbial producers in a desired way that leads to enhanced lactate yields, titers, productivity, and purity. A combination of rational and evolutionary approaches would provide additional flexibility in selecting the most suitable technique for metabolic engineering design that can be applied to the whole genome, multiple pathway genes or a single gene. High-throughput screening of thousands of isolates in short processing times greatly facilitates the discovery of new organisms as promising candidates for industrial production of lactic acid. Future efforts in metabolic engineering will also benefit from the recent progress in genomics, whole genome sequencing, annotation and functional characterization of key metabolic enzymes in re-routing of metabolic flux. Our continuously improving understanding of metabolic pathways and related tools for their effective manipulation together with advances in optimal design of efficient cultivation and recovery systems is expected to boost lactic acid production to levels that can adequately respond to the growing multi-billion dollar market for this important chemical commodity. Many of the metabolic engineering challenges and opportunities related to lactic acid could be relevant in the construction of metabolic pathways in platform cell factories for the production of other metabolites and building block chemicals of industrial importance. References 1 Abdel-Rahman, M.A. et al. (2011) Isolation and characterisation of lactic acid bacterium for effective fermentation of cellobiose into optically pure homo L-(+)-lactic acid. Appl. Microbiol. Biotechnol. 89, 1039–1049 2 Wee, Y.J. et al. (2006) Batch and repeated batch production of L(+)lactic acid by Enterococcus faecalis RKY1 using wood hydrolyzate and corn steep liquor. J. Ind. Microbiol. Biotechnol. 33, 431–435 3 Singhvi, M. et al. (2010) D-(-)-lactic acid production from cellobiose and cellulose by Lactobacillus lactis mutant RM2-24. Green Chem. 12, 1106–1109 4 Rafael, A. et al., eds (2010) Poly(lactic acid) Synthesis, Structures, Properties, Processing, and Applications, Wiley 5 Gao, C. et al. (2011) Biotechnological routes based on lactic acid production from biomass. Biotechnol. Adv. 29, 930–939 6 Christopher, L.P. et al. (2014) Draft genome sequence of a new homofermentative, lactic acid-producing Enterococcus faecalis isolate, CBRD01. Genome Announc. 2, e00147–e214 7 Madhavan Nampoothiri, K. et al. (2010) An overview of the recent developments in polylactide (PLA) research. Bioresour. Technol. 101, 8493–8501 8 Subramanian, M.R. et al. (2014) Production of lactic acid using a new homofermentative Enterococcus faecalis isolate. Microb. Biotechnol. http://dx.doi.org/10.1111/1751-7915.12133. Published online June 3, 2014 9 So¨derga˚rd, A. and Stolt, M. (2002) Properties of lactic acid based polymers and their correlation with composition. Prog. Polym. Sci. 27, 1123–1163 10 Amass, W. et al. (1998) A review of biodegradable polymers: uses, current developments in the synthesis and characterization of biodegradable polyesters, blends of biodegradable polymers and recent advances in biodegradation studies. Polym. Int. 47, 89–144 11 Martinez, F.A.C. et al. (2013) Lactic acid properties, applications and production: A review. Trends Food Sci. Technol. 30, 70–83 12 Ramı´rez-Lo´pez, C.A. et al. (2011) Chemicals from biomass: synthesis of lactic acid by alkaline hydrothermal conversion of sorbitol. J. Chem. Technol. Biotechnol. 86, 867–874 13 Taskila, S. and Ojamo, H. (2013) The current status and future expectations in industrial production of lactic acid by lactic acid bacteria. In Lactic Acid Bacteria - R & D for Food, Health and Livestock Purposes (Kongo, M., ed.), pp. 615–632, InTech 643
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39 Shinkawa, S. et al. (2011) Improved homo L-lactic acid fermentation from xylose by abolishment of the phosphoketolase pathway and enhancement of the pentose phosphate pathway in genetically modified xylose-assimilating Lactococcus lactis. Appl. Microbiol. Biotechnol. 91, 1537–1544 40 Yang, X. et al. (2013) Efficient production of L-lactic acid by an engineered Thermoanaerobacterium aotearoense with broad substrate specificity. Biotechnol. Biofuels 6, 124 41 Angermayr, S.A. et al. (2012) Engineering a cyanobacterial cell factory for production of lactic acid. Appl. Environ. Microbiol. 78, 7098–7106 42 Varman, A. et al. (2013) Photoautotrophic production of D-lactic acid in an engineered cyanobacterium. Microb. Cell Fact. 12, 117 43 Wang, Q. et al. (2011) Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose. Proc. Natl. Acad. Sci. U.S.A. 108, 18920–18925 44 Hofvendahl, K. and Hahn–Ha¨gerdal, B. (2000) Factors affecting the fermentative lactic acid production from renewable resources. Enzyme Microb. Technol. 26, 87–107 45 Papagianni, M. and Avramidis, N. (2011) Lactococcus lactis as a cell factory: A twofold increase in phosphofructokinase activity results in a proportional increase in specific rates of glucose uptake and lactate formation. Enzyme Microb. Technol. 49, 197–202 46 Ge, X.Y. et al. (2011) Improvement of L-lactic acid production by osmotic-tolerant mutant of Lactobacillus casei at high temperature. Appl. Microbiol. Biotechnol. 89, 73–78 47 Yu, L. et al. (2008) Genome shuffling enhanced L-lactic acid production by improving glucose tolerance of Lactobacillus rhamnosus. J. Biotechnol. 134, 154–159 48 Wu, C. et al. (2012) A combined physiological and proteomic approach to reveal lactic-acid-induced alterations in Lactobacillus casei Zhang and its mutant with enhanced lactic acid tolerance. Appl. Microbiol. Biotechnol. 93, 707–722 49 Zhao, J. et al. (2013) Homofermentative production of optically pure Llactic acid from xylose by genetically engineered Escherichia coli B. Microb. Cell Fact. 12, 57 50 Okano, K. et al. (2009) Efficient production of optically pure Dlactic acid from raw corn starch by using a genetically modified Llactate dehydrogenase gene-deficient and alpha-amylase-secreting Lactobacillus plantarum strain. Appl. Environ. Microbiol. 75, 462–467 51 Yoshida, S. et al. (2011) Homo-D-lactic acid production from mixed sugars using xylose-assimilating operon-integrated Lactobacillus plantarum. Appl. Microbiol. Biotechnol. 92, 67–76 52 Tamakawa, H. et al. (2012) Efficient production of L-lactic acid from xylose by a recombinant Candida utilis strain. J. Biosci. Bioeng. 113, 73–75 53 Hu, C-Y. et al. (2011) The direct conversion of xylan to lactic acid by Lactobacillus brevis transformed with a xylanase gene. Green Chem. 13, 1729–1734 54 Zhang, J. et al. (2012) Enhanced acid tolerance in Lactobacillus casei by adaptive evolution and compared stress response during acid stress. Biotechnol. Bioprocess Eng. 17, 283–289 55 Triratna, L. et al. (2011) Genome-shuffling-improved acid tolerance and lactic acid production in Lactobacillus plantarum for commercialization. Microbiol. Indones. 5, 21–26 56 Kadam, S.R. et al. (2006) Strain improvement of Lactobacillus delbrueckii NCIM 2365 for lactic acid production. Process Biochem. 41, 120–126 57 John, R. and Madhavan Nampoothiri, K. (2008) Strain improvement of Lactobacillus delbrueckii using nitrous acid mutation for L-lactic acid production. World J. Microbiol. Biotechnol. 24, 3105–3109 58 Bai, D-M. et al. (2004) Strain improvement of Rhizopus oryzae for overproduction of L(+)-lactic acid and metabolic flux analysis of mutants. Biochem. Eng. J. 18, 41–48 59 Cobb, R.E. et al. (2013) Directed evolution as a powerful synthetic biology tool. Methods 60, 81–90 60 Nevoigt, E. (2008) Progress in metabolic engineering of Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 72, 379–412 61 Luhe, A.L. et al. (2011) Increase of ethanol tolerance of Saccharomyces cerevisiae by error-prone whole genome amplification. Biotechnol. Lett. 33, 1007–1011
Metabolic engineering as a tool for enhanced lactic acid production Bikram P. Upadhyaya1,2, Linda C. DeVeaux1,2,3, and Lew P. Christopher1,2 1
Center for Bioprocessing Research & Development, South Dakota School of Mines & Technology, Rapid City 57701, SD, USA Biomedical Engineering Program, South Dakota School of Mines & Technology, Rapid City 57701, SD, USA 3 Department of Chemistry & Applied Biological Sciences, South Dakota School of Mines & Technology, Rapid City 57701, SD, USA 2
Metabolic engineering is a powerful biotechnological tool that finds, among others, increased use in constructing microbial strains for higher lactic acid productivity, lower costs and reduced pollution. Engineering the metabolic pathways has concentrated on improving the lactic acid fermentation parameters, enhancing the acid tolerance of production organisms and their abilities to utilize a broad range of substrates, including fermentable biomass-derived sugars. Recent efforts have focused on metabolic engineering of lactic acid bacteria as they produce high yields and have a small genome size that facilitates their genetic manipulation. We summarize here the current trends in metabolic engineering techniques and strategies for manipulating lactic acid producing organisms developed to address and overcome major challenges in the lactic acid production process. Importance of lactic acid Lactic acid, or 2-hydroxypropanoic acid, is a naturally occurring chiral organic compound that has been used in both the food and non-food industries for almost 120 years [1]. Lactic acid is approved by the U.S. Food and Drug Administration as GRAS (generally regarded as safe), so its applications in food and other chemical industries are diverse [2]. Cosmetic industries also benefit from lactic acid, as it is used in the manufacture of hygiene and esthetic products [3]. Furthermore, lactic acid has gained tremendous demand as feedstock for the chemical synthesis of poly-lactic acid (PLA) [4]. Since PLA is biodegradable, it can be utilized as an environmentally friendly alternative to petro-chemically derived plastics in the textile, medical, and pharmaceutical industries [5]. The current market price for 88% food grade lactic acid is $1400– 1600 per metric ton (see: http://www.icis.com/chemicals/ channel-info-chemicals-a-z/), with an annual demand of 130 000–150 000 metric tons, which is estimated to reach 367 300 metric tons by 2017 (http://www.prweb.com/ releases/lactic_acid/polylactic_acid/prweb9369473.htm), and over one million tons by 2020 [6]. The rapid annual Corresponding author: Christopher, L.P. ([email protected]). Keywords: metabolic engineering; lactic acid production; lactic acid purity; acid tolerance; carbon source; fermentation parameters. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.10.005
growth of approximately 20% is expected to come from the increasing demand of PLA as a biodegradable polymer. In this review, we discuss recent progress in metabolic engineering strategies, based on both evolutionary and rational methodologies, which have been effective in improving lactic acid production and efficiency in a variety of microorganisms (Figure 1). Challenges and metabolic engineering solutions for lactic acid production Based on a PLA price of $2.2/kg and factoring in the plant size, raw material cost, and capital investment, a target manufacturing cost of lactic acid has been estimated at $0.55/kg [7]. However, in order to minimize production costs, further biotechnological advances are needed to develop high-performance lactic acid producing microorganisms that enhance the fermentation and recovery efficiency in lactic acid production. Lactic acid bacteria (LAB) naturally produce lactic acid as the primary metabolic endproduct, but the chemical and optical purity can vary, and these organisms are generally fastidious, requiring costly nutritional supplementation. Lactobacillus (Lb.) sp. and Lactococcus (L.) sp. are the predominant species used for lactic acid production, but we have recently reported a high yield lactic acid producing Enterococcus isolate that is a promising candidate for metabolic engineering, and does not have the complex nutritional requirements of other LAB [6,8]. Because of their innate acid tolerance, yeasts such as Candida sp. are being developed for lactic acid production, but also require metabolic engineering to introduce and optimize pathways. We discuss here the potential of metabolic engineering as a successful tool to improve the lactic acid fermentation process in a cost-efficient and environmentally friendly way. Emphasis is given to the four major challenges associated with lactic acid production that can be addressed by employing suitable metabolic engineering strategies: (i) purity, (ii) acid tolerance, (iii) carbon source, and (iv) industrial parameters. Challenge 1: Purity of lactic acid Optically and chemically pure lactic acid is required for PLA synthesis; optical purity is a major factor that determines the physical properties of PLA. PLA with a high crystallinity and a high melting point, suitable for the production of fibers, oriented films and liquid crystals [9], could be produced from either the optically pure Trends in Biotechnology, December 2014, Vol. 32, No. 12
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Metabolic engineering
Raonal Engineering
Evoluonary engineering
Pathway redirecon
Classical strain improvement
Increased gene expression
Shotgun mutagenesis approaches
Genome shuffling
Heterologous gene expression
Whole genome amplificaon
Mulplex automated genomic engineering TRENDS in Biotechnology
Figure 1. Metabolic engineering approaches used to address challenges in lactic acid production.
L(+)- or the D( )-lactic acid isomer, but not from a racemic mixture of the two isomers. A main advantage of microbial fermentation processes [10,11] over chemical production methods [12] is the ability to produce optically pure lactic acid. However, the optical purity of lactic acid is dependent on the microbial strain utilized, and can be further optimized. The chemical purity of lactic acid is affected by the composition of the fermentation medium, which in turn is a function of the nutritional requirements of the microbial producer. Traditionally, industrial lactic acid has been produced by fastidious LAB such as Lactobacillus, which
Lignocellulosic feedstock Glucose
(A)
(B)
PEP
Fumarate NADH
Acetyl-CoA
Pyruvate
Ethanol
DH )-L L(+
Succinate
NADH NADH
H
ATP
LD
2NAD+
NAD+
-)-
ADP
D(
2NADH
Acetate NAD+
L(+)-Lactate
NAD+
D(-)-Lactate TRENDS in Biotechnology
Figure 2. Schematic representation of the carbon metabolic pathways in lactic acid fermentation. Broken arrows represent multiple consecutive steps in the pathways. Unshaded area (desired route): lactic acid metabolic pathway. Shaded area (unwanted route of co-product formation): (A) ethanol/acetate metabolic pathway in LAB; (B) succinate metabolic pathway in Escherichia coli.
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utilize expensive carbon (glucose) and nitrogen (amino acids) sources together with vitamins and minerals [13]. Most LAB lack the ability to synthesize growth factors, and generally have complex nutritional requirements, which results in increased purification costs of the final product. As purification costs comprise a significant portion of the overall production costs, the purity of food grade (80–90% purity) and pharmaceutical grade (>90%) lactic acid significantly influences its price [14]. Metabolic engineering solutions to Challenge 1 The laboratory model Escherichia coli has been used in a number of applications due to its ease of growth and well established methods of molecular and genetic manipulations [15–18]. However, E. coli requires metabolic engineering to direct pyruvate toward the single fermentation product of lactic acid (Figure 2). Genes encoding enzymes catalyzing conversion of pyruvate to succinate, acetate, and ethanol have been inactivated, resulting in higher production and chemical purity of the desired lactate product (Table 1). LAB have been engineered to increase the optical and chemical purity of lactic acid. Lb. helveticus in its native state produces a racemic mixture of L(+)- and D( )-lactic acid isomers. By removing the promoter region of the gene for D( )-lactate dehydrogenase (LDH), Lb. helveticus produces only the L(+)-isomer [19]. However, the complex nutritional requirements of the LAB complicate industrial processes and increase costs. To circumvent this, Corynebacterium glutamicum, which can produce lactic acid in minimal medium, has been engineered to express the D( )LDH gene from L. bulgaricus, producing D( )-lactate with >99.9% optical purity [20].
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Table 1. Major challengesa addressed utilizing metabolic engineering tools for enhanced lactic acid production Approach Rational engineering
Major challenge addressed Purity
Acid tolerance Carbon source
Evolutionary engineering
Acid tolerance
Carbon source
Outcome(s) Pure L(+)-lactic acid
Organisms Lactobacillus (Lb.) helveticus
Refs [19]
Pure L(+)-lactic acid (>99%) Titer – 97 g/l, Yield – 90.0%, Productivity – 3.17 g/l/h (Sucrose fermentation) Pure (99.9%) D(S)-lactic acid Titer – 17.92 g/l Ethanol-free lactic acid Titer – 92 g/l, Yield – 94.0% Pure (99.3%) L(+)-lactic acid Yield – 93.0% Pure (99.9%) L(+)-lactic acid Titer – 122 g/l pH 3.0: increased survival after 2 h Cell survival at pH 3.3 Xylose to produce L(+)-lactic acid Purity – 99.5%, Titer – 62.0 g/l Yield – 97.0%, Productivity – 1.631 g/l/h (Xylose fermentation) Glycerol to produce D(S)-lactic acid Purity – 99.97%, Titer – 100.3 g/l Yield – 78.0%, Productivity – 2.78 g/l/h (Glycerol fermentation) Glycerol to produce L(+) lactic acid Purity – 99.9%, Titer – 50 g/l Yield – 93.0%, Productivity – 1.3 g/l/h (Glycerol fermentation) Xylose to produce L(+)-lactic acid Purity – 99.6%, Titer – 50.1 g/l, Yield – 94.6% (Xylose fermentation) Corn starch to produce D(S)-lactic acid Purity – 99.6%, Titer – 73.2 g/l Yield – 0.85 g per g of consumed sugar Productivity – 3.86 g/l/h (Starch fermentation) Mixture of glucose and xylose to produce D(S)-lactic acid Purity – 99.5%, Titer – 74.2 g/l, Yield – 0.78 g per g of consumed sugar, Productivity – 5.6 g/l/h (25 g/l xylose + 75 g/l glucose fermentation) CO2 to produce D(S)-lactic acid Titer – 1.14 g/l (photoautotrophic condition) Xylose to produce L(+)-lactic acid Purity – 99.9%, Titer – 93.9 g/l, Yield – 91.0%, Productivity – 2.18 g/l/h (Xylose fermentation) Xylan to produce lactic acid Titer – 1.7 g/l (Xylan fermentation) At pH 4: titer increased 3-fold over wild type Cell survival at pH 4.3 Titer – Increased by 13.6% Improved cell activity at pH 3.8 Titer – 3.36 g/l, Yield – 95%, Productivity 2.25 g/l/ h At pH 4: 64.5% improvement in lactic acid titer over the wild type Titer – 12 g/l At pH 3.8: 2.6-fold increase in cell growth; Titer - 3.1-fold increase, Productivity – 5.770.05 g/l/h Xylose to produce L(+)-lactic acid Purity – 99.5%, Titer – 62.0 g/l, Yield – 97.0%, Productivity – 1.631 g/l/h (Xylose fermentation) Sucrose to produce D(S)-lactic acid Titer – 110 g/l, Yield – 73.0% Sugarcane molasses and corn steep liquor to produce L(+)-lactic acid
Escherichia coli
[18]
Corynebacterium glutamicum
[20]
Candida (C.) sonorensis
[27]
Thermoanaerobacterium aotearoense Saccharomyces cerevisiae Lactococcus (L.) lactis Lb. casei E. coli
[40] [28] [26] [48] [49]
E. coli
[17]
E. coli
[16]
L. lactis
[39]
Lb. plantarum
[50]
Lb. plantarum
[51]
Synechocystis sp.
[42]
C. utilis
[52]
Lb. brevis
[53]
Lactobacillus Lb. casei
[23] [54]
Lb. pentosus
[32]
Lb. plantarum
[55]
Lb. rhamnosus
[31]
E. coli
[49]
Lb. lactis
[24]
E. coli
[18]
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Table 1 (Continued ) Approach
Major challenge addressed
Parameters
Outcome(s) Purity > 99.0%, Titer – 75 g/l, Yield – 85.0%, Productivity – 1.18 g/l/h Cellobiose and cellulose to produce D(S)-lactic acid Titer – 80 g/l (cellobiose), 73 g/l (cellulose) Productivity – 1.66 g/l/h (cellobiose), 1.52 g/l/h (cellulose) Sucrose to produce lactic acid Titer – 135 g/l, Yield – 90.0% Starch to produce lactic acid Titer – 54 g/l Starch to produce L(+)-lactic acid Purity – 99.05%, Titer – 79.4 g/l Reduced substrate (glucose) inhibition Purity – 99.0%, Titer – 184 g/l, Yield > 90.0%
Organisms
Refs
Lb. lactis
[3]
Lb. delbrueckii
[56]
Lb. delbrueckii
[57]
Rhizopus oryzae
[58]
Lb. rhamnosus
[47]
a
Information on the main challenge addressed is provided in bold; additional outcome(s) are presented, where available.
Challenge 2: Acid tolerance of lactic acid producing organisms Glucose fermentation by homofermentative LAB requires slightly acidic to neutral pH, as low pH has an inhibitory effect on cellular metabolism, and thus lactic acid production. The majority of LAB cannot grow below pH 4, although the pKa of lactic acid is 3.78. In order to maintain a neutral pH for cell survival, lime is routinely added to the fermentors, which results in more than 90% of the lactic acid present as calcium lactate. Following fermentation, the calcium lactate-containing broth is acidified with sulfuric acid to produce lactic acid and insoluble calcium sulfate [21]. The disadvantages of this process include the production of a large amount of calcium sulfate (gypsum) as a by-product and a high sulfuric acid consumption causing corrosion problems [22]. The amount of gypsum is usually higher than the amount of lactic acid produced, and more than a ton of gypsum is generated for every ton of lactic acid, presenting a waste disposal problem and a major cost factor in the product recovery stage of commercial operations. Ideally, microbial fermentation would occur in medium with a pH at or below the pKa of lactic acid, allowing direct purification of the acid form. Metabolic engineering solutions to Challenge 2 Classical mutagenesis (Box 1) has been applied to select for variants of Lactobacillus sp. with increased tolerance to the acidified medium produced during fermentation. After UV and nitrosoguanidine treatment, these improved strains are capable of lactic acid production at rates and yields similar to those of the traditional, neutral-pH lactic acid processes [23,24]. Acidic conditions represent an environmental stress that activates specific and global stress responses, involving induction of the synthesis of several proteins [25]. In order to increase resistance to the acidic conditions resulting from lactic acid fermentation, enzymes such as trehalose 6-phosphate phosphatase from Propionibacterium freudenreichii [25] has been expressed in L. lactic resulting in 5- to 10-fold higher survivability at pH 3.0. In a similar study, the histidine decarboxylation pathway from Streptococcus thermophilus [26] was expressed in L. lactis, resulting in survival at pH levels as low as 3 in which the host cells were rapidly dying. 640
To capitalize on the natural acid resistance of yeasts, lactic acid productivity has been improved by introducing the gene encoding L(+)-LDH from heterologous sources. The bovine gene encoding LDH has been successfully expressed in both Candida (C.) utilis and Saccharomyces cerevisiae, and the gene encoding LDH from Lb. helveticus has been expressed in Candida sonorensis [27–29]. Genome shuffling (Box 1) has been a successful technique to enhance complex phenotypes while reducing the deleterious effects of accumulating extraneous mutations [23,30]. In Lactobacillus, this technique was used to lower the optimal pH of lactic acid production, without loss of yield. One population was adapted gradually to fermentation in medium with incrementally lower pH, while a second population was mutagenized with nitrosoguanidine, then selected on medium with lowered pH. Following protoplast formation, the two populations were fused. Fused isolates were selected on low pH medium and screened for high lactic acid productivity [23,31]. This resulted in strains that produced three times the wild type levels of lactic acid at pH 3.8, close to the pKa of lactic acid. Recently, whole genome amplification (Box 1), a new method for introducing random mutations using PCR and electroporation, has been used to improve the acid tolerance of Lb. pentosus, producing a strain with 95% lactic acid yield at pH 3.8, in which the wild type strain did not grow [32]. Since this technique does not rely on protoplast fusion, it could have a wider application to other species beyond the Lactobacilli. Challenge 3: Carbon source for lactic acid production As polymer producers and other industrial users usually require large quantities of lactic acid at a relatively low cost, inexpensive raw materials are essential for the economic feasibility of the microbial process. The use of starch and refined sugars (glucose) as feedstock increase the cost of lactic acid, as in a typical lactic acid fermentation process the raw material cost constitutes between 40% and 70% of the total production cost [33]. The raw materials for industrial lactic acid production need to have several characteristics, such as low cost, low levels of contaminants, rapid fermentation rate, high lactic acid yields, little or no by-product formation, and year-round availability. Since glucose or any other food-based feedstock directly compete
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Box 1. Evolutionary engineering Evolutionary engineering resembles Darwinian evolution, but with much shorter time, and thus faster progression toward a desired characteristic [59]. A very small subset of the population will have been changed, and it is unnecessary to understand the molecular or genetic mechanism behind the improved characteristics. Because of this, however, it is usually not possible to transfer specific genetic information to other strains [60]. Classical strain improvement. Selection for beneficial mutations through classical strain improvement may rely on spontaneous mutations that occur in any growing population. Alternatively, a mutagen may be applied to increase the frequency of mutations. In either case, the resulting mixed population is subjected to conditions that will select or enrich for those individual cells that have acquired the ability to survive (Figure I). Shotgun mutagenesis approaches. Unlike classical strain improvement, shotgun mutagenesis approaches can introduce many more mutations with a higher throughput, reducing the mutational load on any individual. This shortens the time to carry out the process which, along with higher sensitivity for detection, allows more mutagenized individuals to be assessed in a short time. The methods described below do not represent an exhaustive list, but provide examples of these next-generation approaches (see Figure 1 in main text). Genome shuffling. Genome shuffling utilizes recursive protoplast fusion to allow recombination between genomes so that only the desired mutation(s) may be selected [23]. Briefly, two populations are generated. One population is adapted by selecting for spontaneous mutations and the second population is generated through chemical or physical mutagenesis, creating a library of strains, presumably with many mutations per chromosome. The genomes of the two populations are then ‘‘shuffled’’ by fusing cells from each population, followed by selection for the desired trait (Figure II). Whole genome amplification (WGA). In WGA, short, random oligonucleotide primers are used to prime PCR with the genome of
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the organism as the template. By using a non-proofreading polymerase, fragments are generated with random nucleotide changes introduced. These fragments are then re-introduced into the organism to be modified using electroporation, with subsequent selection or high-throughput screening for the desired traits [32,61]. Multiplex Automated Genome Engineering (MAGE). MAGE has automated the introduction of random, mutated genomic pieces, speeding up directed evolution. Using multiplex DNA synthesis, random mutated pieces are generated and introduced into cells, and, as with other approaches, useful mutants are selected with suitable media or in various environmental conditions.
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Figure II. Schematic representation of heterologous gene expression. Metabolic pathway system of the starting strain converts substrate (X) to intermediate product (I) but lacks the enzymatic pathway to the target product (Z). Expression of the cloned gene from an unrelated strain (capable of converting I to Z) allows the conversion of X to Z in the engineered strain.
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Figure I. Schematic representation of pathway re-direction and increased gene expression (A) Metabolic pathway system of the starting strain converting substrate X to co-product Y and target product Z. (B) Elimination of the enzyme pathways that convert X to Y resulting in increased levels of target product Z. (C) Overexpression of the enzymes responsible for converting X to Z resulting in increased levels of target product Z.
with the food supply chain, there is a growing interest in the use of lignocellulosic biomass (Figure 2) as a renewable and low-cost substrate for the microbial production of lactic acid [34–37]. However, the release of fermentable sugars from the complex lignocellulose polymer requires additional processing steps (e.g., biomass pre-treatment, hydrolysis and possible detoxification of lignocellulosic hydrolyzates), which increase the production cost of lactic acid. Metabolic engineering solutions to Challenge 3 A natural isolate of E. coli was engineered to remove the repressor of genes encoding enzymes required for sucrose utilization, which may allow lactate production from more inexpensive sugar sources [38]. Similarly in L. lactis, optically
pure L(+)-lactic acid was made from xylose, by elimination of the phosphoketolase pathway of xylose utilization [39]. With improved molecular biology techniques, engineering similar deletion mutations in less well-studied organisms has been effective. Thermoanaerobacterium aotearoense, a thermophile and obligate anaerobe that can convert lignocellulose directly to L(+)-lactate, was made homo-fermentative by blocking the competing formation of acetic acid [40]. Microalgae are being considered for lactic acid production, as, among other reasons, the utilization of light energy to fix carbon could potentially eliminate feedstock costs. Synechocystis sp. have been engineered (Table 1) to express either an L(+)-LDH gene to produce L(+)lactate [41] or a mutated glycerol dehydrogenase gene 641
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Box 2. Rational metabolic engineering A rational approach to metabolic engineering refers to a directed, and usually very specific, alteration of a known pathway or protein, which is expected to have a particular outcome. As such, it requires extensive genetic and metabolic information about the target organism. Usually these directed changes yield the predicted result. However, rational engineering perturbs normal metabolism and has also activated novel, sometimes cryptic pathways, revealing previously unrecognized complex cellular regulatory networks [60]. In addition, when strong selective pressures are applied, compensatory mutations present in the population may alter other existing pathways, for example, by changing substrate specificities of related enzymes [43]. Pathway re-direction. Generally, pathway re-direction involves genetic modification to eliminate competing pathways for pyruvate utilization or production, or pathways leading to accumulation of unwanted metabolites (Figure I). In addition to requiring knowledge of the existing pathways, the organism must be amenable to such manipulations. Increasingly, pathway re-direction is combined with other molecular strain improvements to optimize flux through the desired pathway. It has also been a successful approach to improve the optical purity of lactic acid. For example, Escherichia coli contains genes for production of both the D( )- and L(+)-isomers. The optical purity was increased by elimination of the pathways for the alternate isomer, or by preventing undesired utilization of the desired isomer [16]. Increased gene expression. A complementary approach to pathway re-direction is to increase the transcription of the gene(s) encoding the enzyme(s) in the desired pathway (Figure I). Increases in gene expression are most easily accomplished by cloning the gene(s) of interest, and expressing on a multicopy, extra-chromosomal element within the cell, or by replacing a native promoter with a more efficient promoter on the chromosome. Heterologous gene expression. Organisms with desirable properties for lactic acid production, but lacking key enzymatic processes, can be engineered to express non-native genes, introducing novel metabolic characteristics that improve their industrial importance (Figure II). While advances in molecular biology techniques have broadened the application of such heterologous protein expression, there is no single system that works across the board.
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Figure I. Schematic representation of classical strain improvement. Arrows represent selection of the desired strains. Broken squares refer to selected strains for the next round of mutagenesis. Ovals represent individuals within the population. Plus (+) represents improved characteristics; minus (–) represents undesirable characteristics.
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Figure II. Schematic representation of genome shuffling as a metabolic engineering tool for strain improvement.
to produce D( )-lactate [42], both originating from Bacillus sp. [43].
repression [44]. These parameters depend on the type of microbial producer and its metabolic pathways (Figure 2).
Challenge 4: Parameters for industrial lactic acid production The key economic drivers for a cost-efficient lactic acid production are: lactate yield (% of theoretical maximum from carbon source), volumetric productivity (g/l/h), and titer (g/l). The commercial production process requires the use of non-fastidious and robust microorganisms that can meet the industrial needs for high yield, productivity and titer of lactic acid of at least 80%, 2.5 g/l/h and 100 g/l, respectively [8]. Other important fermentation parameters include specific growth rate of the production organism, metabolite formation, ability to ferment both pentose and hexose sugars, no substrate or end-product inhibition, resistance to inhibitory compounds formed during pre-treatment of lignocellulosic biomass, and minimizing carbon catabolite
Metabolic engineering solutions to Challenge 4 As naturally high lactic acid producers, LAB such as Lactococci and Lactobacilli have been engineered to heterologously express other enzymes that improve productivity. L. lactis expressing part of the gene encoding phosphofructokinase from Aspergillus niger showed increased flux through the glycolytic pathway, resulting in increased lactate production [45]. Within the species, L. lactis IL with a disrupted phosphoketolase pathway, but enhanced pentose phosphate pathway due to expression of the transketolase genes from L. lactis IO-1, produced L(+)-lactic acid from xylose at levels close to the theoretical value [39]. Recently, Lb. helveticus was engineered to express only L(+)-lactate by deletion of the gene for D( )-LDH, and an additional copy of the gene for
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Review L(+)-LDH was inserted in its place, resulting in almost twice the total native production, and all optically pure L(+)-lactate [19]. In another study, D( )-LDH has been overexpressed in E. coli, which resulted in increased production and yield of D( )-lactate from glycerol [17]. Eukaryotic lactic acid producers, such as Candida sp., have also been engineered for the production of L(+)-lactic acid. The gene encoding the pyruvate decarboxylase of C. utilis was disrupted, which, together with heterologous expression of LDH made L(+)-lactic acid with high efficiency [29]. Genome shuffling has been used to increase the tolerance to high substrate concentrations, which is normally inhibitory to cell growth and lactic acid production [46]. Simultaneous selection for glucose tolerance and high lactic acid production yielded a strain of Lb. rhamnosus that made 70% more lactic acid than the wild type at 150 g/l glucose [47]. Critical analysis of metabolic engineering advances Acid stress and end-product inhibition are still among the major bottlenecks in commercial lactic acid production; hence, further progress in metabolic modeling and systems biology would help engineer advanced biosynthetic pathways for an improved biotechnological process. Furthermore, the successful identification of genes and proteins associated with stress response and tolerance would facilitate the design of rational approaches for metabolic engineering (Box 2). In addition, the acid tolerance and carbon substrate range of producing organisms can be broadened, which allows the design of a sustainable and environmentally sound production process. More focus should be given to the heterologous expression of enzyme and metabolic pathways in yeasts and fungi as they are more acid tolerant than LAB and naturally produce more optically pure lactic acid. As the raw material is a major cost factor in lactic acid production, future developments in metabolic engineering are expected to enhance the utilization of lignocellulosic biomass (e.g., biomass-derived hexose and pentose sugars) and biomass-derived waste streams (e.g., carbon dioxide, paper sludge, agriwaste, and food waste). In the case of lignocellulosic biomass, efforts should be made to increase microbial resistance toward inhibitory compounds formed during biomass pre-treatment and pentose fermentation, and toward carbon catabolite repression caused by heterologous sugar substrates present in biomass. However, waste streams derived from biomass would likely be the preferred feedstock for lactic acid production as (i) it eliminates the need for biomass pre-treatment and formation of unwanted inhibitory products utilizing a low-cost feedstock thereby minimizing total cost; and (ii) it utilizes waste by-product thereby alleviating pollution and greenhouse gas emission problems. From this perspective, engineering microalgae and cyanobacteria to increase their photosynthetic efficiency of lactic acid production from carbon dioxide could significantly lower production costs. Concluding remarks and future perspectives The rapidly growing demand for lactic acid dictates the need for use of robust and highly efficient microorganisms that meet the strict industrial requirements for cost-efficient lactic acid production. To address this challenge, metabolic engineering is increasingly employed by biotechnologists
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as a powerful tool that can manipulate the traits of lactic acid microbial producers in a desired way that leads to enhanced lactate yields, titers, productivity, and purity. A combination of rational and evolutionary approaches would provide additional flexibility in selecting the most suitable technique for metabolic engineering design that can be applied to the whole genome, multiple pathway genes or a single gene. High-throughput screening of thousands of isolates in short processing times greatly facilitates the discovery of new organisms as promising candidates for industrial production of lactic acid. Future efforts in metabolic engineering will also benefit from the recent progress in genomics, whole genome sequencing, annotation and functional characterization of key metabolic enzymes in re-routing of metabolic flux. Our continuously improving understanding of metabolic pathways and related tools for their effective manipulation together with advances in optimal design of efficient cultivation and recovery systems is expected to boost lactic acid production to levels that can adequately respond to the growing multi-billion dollar market for this important chemical commodity. Many of the metabolic engineering challenges and opportunities related to lactic acid could be relevant in the construction of metabolic pathways in platform cell factories for the production of other metabolites and building block chemicals of industrial importance. References 1 Abdel-Rahman, M.A. et al. (2011) Isolation and characterisation of lactic acid bacterium for effective fermentation of cellobiose into optically pure homo L-(+)-lactic acid. Appl. Microbiol. Biotechnol. 89, 1039–1049 2 Wee, Y.J. et al. (2006) Batch and repeated batch production of L(+)lactic acid by Enterococcus faecalis RKY1 using wood hydrolyzate and corn steep liquor. J. Ind. Microbiol. Biotechnol. 33, 431–435 3 Singhvi, M. et al. (2010) D-(-)-lactic acid production from cellobiose and cellulose by Lactobacillus lactis mutant RM2-24. Green Chem. 12, 1106–1109 4 Rafael, A. et al., eds (2010) Poly(lactic acid) Synthesis, Structures, Properties, Processing, and Applications, Wiley 5 Gao, C. et al. (2011) Biotechnological routes based on lactic acid production from biomass. Biotechnol. Adv. 29, 930–939 6 Christopher, L.P. et al. (2014) Draft genome sequence of a new homofermentative, lactic acid-producing Enterococcus faecalis isolate, CBRD01. Genome Announc. 2, e00147–e214 7 Madhavan Nampoothiri, K. et al. (2010) An overview of the recent developments in polylactide (PLA) research. Bioresour. Technol. 101, 8493–8501 8 Subramanian, M.R. et al. (2014) Production of lactic acid using a new homofermentative Enterococcus faecalis isolate. Microb. Biotechnol. http://dx.doi.org/10.1111/1751-7915.12133. Published online June 3, 2014 9 So¨derga˚rd, A. and Stolt, M. (2002) Properties of lactic acid based polymers and their correlation with composition. Prog. Polym. Sci. 27, 1123–1163 10 Amass, W. et al. (1998) A review of biodegradable polymers: uses, current developments in the synthesis and characterization of biodegradable polyesters, blends of biodegradable polymers and recent advances in biodegradation studies. Polym. Int. 47, 89–144 11 Martinez, F.A.C. et al. (2013) Lactic acid properties, applications and production: A review. Trends Food Sci. Technol. 30, 70–83 12 Ramı´rez-Lo´pez, C.A. et al. (2011) Chemicals from biomass: synthesis of lactic acid by alkaline hydrothermal conversion of sorbitol. J. Chem. Technol. Biotechnol. 86, 867–874 13 Taskila, S. and Ojamo, H. (2013) The current status and future expectations in industrial production of lactic acid by lactic acid bacteria. In Lactic Acid Bacteria - R & D for Food, Health and Livestock Purposes (Kongo, M., ed.), pp. 615–632, InTech 643
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