Production of ethanol from starch-based crops such as wheat and corn is a well-known technology. Such processes have been optimized over a long time and are reaching a level of maturity where further cost reductions, based on improvements in conversion technology, are becoming more difficult. In contrast, processes using lignocellulosic raw materials are still under develop­ment and significant reductions in ethanol production cost can be expected.

With some modifications it is possible to make a basic evaluation of a starch-based process (Fig. 1) and compare it with a process based on ligno­cellulosic material (Fig. 3). This was done by Wingren [17]. The purpose of the evaluation was not to determine the absolute ethanol production cost, but to compare the processes using the same fundamental cost basis and the same assumptions in the investment analysis. A comparison of this kind provides valuable information on the major differences between a commercial process and a process under development.

Both plants were designed for an annual ethanol production of 55 000 m3, which is a rather small plant. This value is on a pure ethanol basis, although the actual distillate was assumed to be 94% (w/w), i. e. no dehydration step was included as this would have been the same in both cases. Also, no off­sites, e. g. production of heat and electricity, were included, only the pure ethanol production facility. In the evaluation no credit was given for carbon dioxide. The cost of the enzymes in a starch-based plant is lower than in a lig- nocellulosic plant. In the study it was assumed to be 0.014 US$ L-1 ethanol, which is slightly higher than the cost reported for the enzymes in a corn — based plant located in the USA [18].

The raw material flow is higher in the lignocellulosic process, 200 000, compared to 126 500, dry metric tons y-1 for the starch-based plant, due to the lower overall ethanol yield and the somewhat lower amount of fermentable sugars in the raw material. The overall energy demand in the lignocellulosic process was estimated to be 16 MJ L-1 ethanol compared to 10 MJ L-1 for the wheat-based process. The fixed capital investment was estimated to be 99 and 53 million US$ for the lignocellulosic and the starch-based processes, respec­tively. A breakdown of costs is presented in Table 3. The estimated ethanol production cost was 0.60 and 0.58 US$ L-1 for the lignocellulosic and starch — based processes, respectively. Major differences were found in the cost of raw material, enzymes, capital, steam as well as income from the co-products. It should be noted that, although significantly higher than in the starch-based process, the enzyme cost in the lignocellulosic process was based on a pro­jected future cost. In the starch-based process the cost of the raw material constitutes as much as 65% of the total production cost. This is typically the case for well-established, mature processes. Thus, the economics of a starch — based process is very dependent on the cost of feedstock.

The lignocellulosic process is more dependent on the income from the co-products. However, the potential price of the syrup is uncertain since its fuel properties are unknown. At 12.9 US$ MWh-1 the income from this co­product was estimated to be 0.03 US$ L-1. In a scenario where the co-product instead has to be disposed of and cannot be utilized as a fuel, the ethanol production cost for the lignocellulosic process would be 0.63 US$ L-1. The

income from the pellets in the lignocellulosic plant reduces the ethanol pro­duction cost by 0.15 US$ L-1 at 20 US$ MWh-1. As in the case of the syrup, the true price of this co-product will be dependent on its fuel properties.

The results of this comparative study led to some important conclusions regarding potential cost reductions in the lignocellulosic process, compared with the starch-based process. The overall ethanol yield in the lignocellu­losic process evaluated is 68% of the theoretical based on the available glucan and mannan in the raw material, a figure that can probably be increased. In addition, a pentose — and galactose-fermenting organism could increase the ethanol production per unit raw material without increasing the capital cost. This is especially important if the raw material is rich in pentoses, e. g. as in straw or hardwood. A reduction in enzyme loading would also be rewarding provided that the ethanol yield could be maintained. Figure 9 shows a break­down of capital costs together with energy costs for the two processes. The largest difference in costs is seen in the conversion steps and in the evapo­ration step. The pretreatment step in the lignocellulosic process represents around 0.093 US$ L-1 ethanol. This cost is attributed to both a high energy demand and to the high cost of the reactor system. This shows the need to improve pretreatment and/or enzymatic hydrolysis so that less severe pre­treatment is required. The higher cost of the SSF step compared with the fermentation step in the starch-based process is due to the longer residence

time and the lower substrate concentration in the lignocellulosic process. An increase in substrate load and productivity in the lignocellulosic process would reduce this difference. The difference in cost between the starch-based process and the lignocellulosic process in the downstream processing steps (evaporation and distillation) would also be reduced if the ethanol concentra­tion in the SSF step could be increased.

3.3

As discussed earlier, the complete genome sequence of Z. mobilis ZM4 has been reported recently [12] following earlier related studies by Korean scien­tists [88-90]. It was found that the genome consists of 2 056416 base pairs forming a circular chromosome with 1998 open reading frames (ORFs) and three ribosomal RNA transcription units. As reported by the authors, “the genome lacks recognizable genes for 6-phosphofructokinase, an essential en­zyme in the Embden-Meyerhof-Parnas pathway, and for two enzymes in the tricarboxylic acid (TCA) pathway, the 2-oxoglutarate complex and malate dehydrogenase. Glucose can be metabolized therefore only by the Entner — Doudoroff pathway”.

Comparison of whole genome microarray data for Z. mobilis ZM1 (ATCC10988) and ZM4 (ATCC 31821) revealed that the 54 ORFs present in ZM4 were absent for ZM1. Four of these ORFs that encode trans­port proteins or permeases, and two that encoded for specific enzymes— NAD(P)H:quinone oxidoreductase and an oxidoreductase related to short — chain alcohol dehydrogenases, were found to be highly expressed in Z. mobilis ZM4. The authors suggested that it is possible these genes relate to the higher specific rates of sugar uptake and ethanol production for ZM4 when compared to ZM1. They also reported that two genes encoding capsular carbohydrate synthesis enzymes were only actively expressed in ZM4 and may contribute to its relatively high resistance to increased osmotic pressure found in high sugar solutions (e. g. in 250-300 g L-1 glucose media).

4

Like E. coli, Klebsiella oxytoca is able to metabolize a variety of biomass — derived monomeric sugars, but unlike E. coli it also has the native ability to transport and metabolize cellulose subunits cellobiose and cellotriose [50, 51]. The PET operon was expressed in K. oxytoca concurrent with the ori­ginal E. coli work [52] and was later chromosomally integrated, resulting in strain P2 [51]. Ethanol production by K. oxytoca P2 from various substrates has been reported (Table 1) [53-56]. K. oxytoca strain BW21, which was de­rived from strain P2 by elimination of the butanediol pathway, produces over 40 g L-1 ethanol in 48 h in OUM1 medium. OUM1 is a medium designed spe­cifically for K. oxytoca, as described below [57].

When Z. mobilis was selected as the source of the PET operon, Z. mo — bilis and Gram-positive Sarcina ventriculi were the only known bacteria with PDC activity [58-61]. Since that time, PDC activity has been identified in other bacteria, including Gram-negative Acetobacter pasteurianus [62] and Zymobacter palmae [3]. The Z. palmae PDC has a higher specificity and lower pyruvate Km than Z. mobilis, S. ventriculi, and A. pasteurianus [63]. The S. ventriculi PDC is different from the Z. mobilis enzyme but is highly related to the PDC found in fungi; its expression in E. coli requires the presence of accessory tRNA due to differences in codon usage [63]. In A. pasteurianus, PDC seems to have the unusual role of functioning in an aerobic pathway, contributing primarily to the conversion of pyruvate to acetaldehyde [62].

The robustness of Gram-positive organisms is appealing for industrial ap­plications, but initial attempts to express the Z. mobilis homoethanol pathway in Bacillus and lactic acid bacteria had limited success [64-67]. However, the discovery of new PDC forms has enabled renewed engineering attempts. The PDCs from S. ventriculi, A. pasteurianus, and Z. mobilis, as well as S. cerevisiae, were each expressed in Bacillus megaterium, with the S. ven — triculi PDC showing the highest activity. When coupled with ADH from Geobacillus stearothermophilus, the S. ventriculi PDC enabled B. megaterium to convert 13.2 gL-1 pyruvate to 3.3 gL-1 ethanol, a tenfold increase rela­tive to strains lacking PDC [68]. The S. ventriculi PDC was also expressed in Lactobacillus plantarum, with production of up to 6 g L-1 ethanol from 40gL-1 glucose [69]. Recent attempts to express the Z. mobilis pathway in Corynebacterium glutamicum have resulted in production of ethanol from glucose [70].

Considerable effort has been extended to engineering of Z. mobilis for im­proved ethanol production, as covered elsewhere in this volume. However, the ethanol titers attained by ethanologenic E. coli LY168 in minimal medium exceed published values for Z. mobilis in rich medium (Table 2).

3

The hydrolysate released from the pretreatment is typically treated with en­zymes in order to break down cellulose and hemicellulose into hexoses and pentoses that are then further fermented to ethanol. Enzyme costs are, how­ever, generally high, so that the search for new enzymes with high efficiency that can be produced at low costs is the key to overcome the bottleneck of this process step [18] (see also Viikari, this volume). Another possible way to reduce treatment costs is to implement recycle loops in order to feed back­washed enzymes into the vessel of enzymatic hydrolysis [19,20]. Enzymes could further be produced at the plant using a stream of the pretreated ma­terial in an on-site enzyme production.

The hydrolysis step is optimized by performing the enzyme treatment to­gether with yeast fermentation of glucose (simultaneous saccharification and fermentation, SSF). The temperature optimum of the enzymes is, however, of­ten higher than the optimum for yeast. This can depress the advantages of SSF compared to separation of the two processes.

4.3

L-Alanine, used in the pharmaceutical industry [150] and as a food addi­tive [151], is commercially produced by enzymatic decarboxylation of L-aspartic acid with either immobilized cells or cells suspensions [152]. How­ever, recent attention has shifted to fermentative production [150,151].

Given our success in producing microbial biocatalysts by a combination of directed engineering and metabolic evolution, we modified lactic-acid produc­ing E. coli B derivative SZ194 for alanine production. The native IdhA gene was replaced with the ribosome binding site, coding region, and transcriptional terminator of the thermostable alanine dehydrogenase alaD from Geobacillus stearothermophilus XL-65-6 (Zhang et al., unpublished results). While the ini­tial microbial biocatalyst was capable of producing l-alanine as the primary fermentation product, long incubation times were required and the productiv­ity was low. As with other microbial biocatalysts designed in our laboratory, metabolic evolution was used for strain improvement. The strain was further engineered to reduce co-product formation (mgsA) and increase the chiral purity (dadX). The final microbial biocatalyst, XZ132, produced near 1.3 M l — alanine from 12%glucose within 48 h, a yield greater than 95%, in AM1 mineral salts media.

6

One approach to reducing the production cost is integration of ethanol pro­duction with another suitable plant, e. g. a combined heat and power plant, a starch-based ethanol plant or a pulp and paper mill. Significant reduction of the production cost was obtained in a study on co-production of ethanol and electricity from softwood, based on conditions in California, USA [44]. One of the benefits is that the syrup or lignin residue can be used for steam pro­duction without prior drying. Another option is to integrate cellulosic ethanol production with starch-based ethanol production to utilize the whole agri­cultural crop. This will increase the production capacity drastically, and it may also help to boost the ethanol concentration resulting from the ligno — cellulosic process, if the ethanol-containing streams can be distilled in the same distillation units. This will have a beneficial effect on the energy de­mands in the distillation and evaporation steps. It might be a disadvantage if the residue cannot be used for animal feed (DDGS). However, it will still have a fuel value, which will help to improve the economics of the overall process. The biorefinery concept is also an interesting option. Using chem­ical and biological transformations, the raw material is processed to produce ethanol and, e. g., modified lignin, specialty chemicals and maybe anaerobic biogas, adding value to the main product. In this case the income from other products improves the overall process economics [45,46].

4

Conclusions

Flowsheeting, combined with estimates of the production cost, is a valuable tool for the comparison of process alternatives and to determine bottlenecks that require further improvement. It is, however, difficult to compare pro­duction costs from different studies due to the many assumptions made in the simulations, such as ethanol yield, productivity and concentration, as no commercial-scale plants are in existence. Also, differences in capacity and cost of raw material, as well as currency exchange rates, add to the uncer­tainty. This is clearly illustrated by the large variation in the estimated ethanol production cost, from 0.13 to 0.81 US$ L-1 ethanol.

The most important parameters for the economic outcome are the feed­stock cost, which varied between 30 and 90 US$ per metric ton, and the plant capacity, which influences the capital cost. It is thus very important to reach a high overall ethanol yield as this is directly related to feedstock and capital costs for a given production capacity.

One of the major research challenges is to improve the hydrolysis of carbo­hydrates through more efficient and less expensive pretreatment methods, but also by enhanced enzymatic hydrolysis with superior enzymes at a reduced enzyme production cost. The latter is one of the most uncertain costs in most economic analyses.

It is also important to achieve a high ethanol concentration in the fer­mentation or SSF steps to reduce the energy demand. This requires new technology for enzymatic hydrolysis (or SSF) at high solids concentrations and the development of robust fermenting organisms that are more toler­ant to inhibitors. They also have to be able to ferment all sugars in the raw material in concentrated hydrolyzates, while maintaining high ethanol pro­ductivity and a high ethanol concentration.

Finally, process integration within the process and with other types of in­dustrial processes, e. g. a combined heat and power plant or a starch-based ethanol plant, will reduce the production cost further. Regarding the immedi­ate future, we believe that these integrated plant concepts will be used in the first successful industrial-scale production of lignocellulosic fuel ethanol.

Adv Biochem Engin/Biotechnol (2007) 108: 329-357 DOI 10.1007/10_2007_059 © Springer-Verlag Berlin Heidelberg Published online: 11 April 2007

Vancouver, British Columbia V6T 1Z4, Canada warren. mabee@ubc. ca

1 Introduction……………………………………………………………………………………………… 330

2 Biofuel Production…………………………………………………………………………………….. 331

2.1 Brazil……………………………………………………………………………………………………….. 333

2.2 United States…………………………………………………………………………………………….. 334

2.3 European Union………………………………………………………………………………………… 337

2.4 Other Biofuel Producing Nations………………………………………………………………… 341

3 Direct Funding Programs in the USA………………………………………………………….. 343

4 Excise Tax Exemptions in the USA…………………………………………………………….. 347

5 Political Goals and Bioethanol-Related Policy………………………………………………. 350

6 Conclusions………………………………………………………………………………………………. 351

References ……………………………………………………………………………………………………. 354

Abstract Biofuels for use in the transportation sector have been produced on a signifi­cant scale since the 1970s, using a variety of technologies. The biofuels widely available today are predominantly sugar — and starch-based bioethanol, and oilseed — and waste oil — based biodiesel, although new technologies under development may allow the use of lignocellulosic feedstocks. Measures to promote the use of biofuels include renewable fuel mandates, tax incentives, and direct funding for capital projects or fleet upgrades. This paper provides a review of the policies behind the successful establishment of the bio­fuel industry in countries around the world. The impact of direct funding programs and excise tax exemptions are examined using the United States as a case study. It is found that the success of five major bioethanol producing states (Illinois, Iowa, Nebraska, South Dakota, and Minnesota) is closely related to the presence of funding designed to support the industry in its start-up phase, while tax exemptions on bioethanol use do not influ­ence the development of production capacity. The study concludes that successful policy interventions can take many forms, but that success is equally dependent upon external factors, which include biomass availability, an active industry, and competitive energy prices.

Keywords Biofuels • Direct funding • Excise tax exemptions • Policy •

Renewable fuel mandates

1

Introduction

Biofuels derived from sustainable biological sources, including agricultural crops, waste vegetable oils, and woody biomass, is advocated by many in­cluding MacLean et al. [1] and McMillan [2] as a potential substitute for petroleum-derived fuels such as gasoline and diesel. The use of biofuels is generally associated with lower greenhouse gas emissions and improved en­ergy balance compared to petroleum-based fuels [3], which makes them an attractive option for combating climate change and meeting national or in­ternational targets of environmental performance. As the biofuel industry is based on agricultural (or potentially forest) biomass, development of the in­dustry will lead to a diversified rural economy and increased employment, which can support domestic development goals [4-6]. The industry has long been promoted as a means to substitute renewable, sustainable biomass for fossil reserves of oil, which may in turn increase the security of energy sup­plies and reduce dependence upon foreign oil [7]. These attributes make biofuel an attractive option for policymakers, offering solutions to a number of domestic challenges. At the same time, policy is needed in order to increase the competitiveness of bio-based fuels, which are generally more expensive to produce than petroleum-based counterparts [8].

Policy options to support biofuel production may take a number of forms. Some options are “top-down” in form, as they are enacted on a national or regional basis and impact all producers and consumers. One such option is the national target, in which policymakers make a public declaration of their intention to meet a certain level of production (often expressed as a percent­age of overall production) in domestic transportation fuel supply. Top-down policy places the emphasis upon governments, which are then responsible for creating an environment supportive towards industrial expansion. The national target should not be confused with a renewable fuels standard (or obligation), which sets legal standards for the minimum levels at which bio­fuels must be blended into transportation fuels. A renewable fuel standard places the emphasis upon industry, who must then meet the renewable fuel standards with their products in order to be eligible for sale. One commonly observed policy option is exemption of biofuels from national excise taxation schemes, which has the effect of reducing producer costs and thus increasing potential profits. This type of incentive can be identified as a subsidy to indus­try, although lower prices can be and are passed to consumers in competitive markets.

Other policy options act in a “bottom-up” fashion, impacting only par­ticular industrial or consumer participants in the biofuel marketplace. One such option is direct government funding of capital projects to increase cap­acity or upgrade distribution networks. Normally, these types of policies are enacted in a competitive fashion, wherein various industrial producers can compete for projects, which are then carried out in conjunction with govern­ment. Another bottom-up type of policy is targeted at increasing biofuel use in government or corporate vehicle fleets.

In some countries, multiple policies covering the range of options de­scribed above have been enacted to support biofuel development (e. g. [9-11]). The presence of multiple policies within these jurisdictions means that de­termining the effectiveness of individual policies is quite difficult. In this paper, implementation of biofuels in several countries is examined. The abil­ity of two measures to promote domestic biofuel production is compared. The first measure considered is exemptions on fuel excise taxes; the second is funding designed to support projects, infrastructure, or capacity develop­ment for bioethanol production. The industry is then evaluated on its ability to successfully promote broad policy goals of employment, environmental performance, and fuel security. A number of recommendations for the for­mulation of future policies are proposed.

2

4.1

Metabolites and Related Products

The production of a range of byproducts from Z. mobilis is reviewed compre­hensively by Johns et al. [6] and Panesar et al. [8] with the former authors identifying potential commercial opportunities for the following products: fructose (using sucrose and a fructokinase negative mutant), sorbitol and glu­conic acid, levan (a fructose polymer), fructo-oligosaccharides and various enzymes. As pointed out in a review by Scopes [91], Z. mobilis is a rich source (on an enzyme content per g cell basis) of many of the enzymes currently used in diagnostic analysis and research. Interestingly, in other studies by Park et al. [92], it was established that the activities of some of the key ED enzymes (e. g. glucokinase, G-6-phosphate dehydrogenase) were unaffected by the relatively high ethanol concentrations produced during fermentation, while the activity of an enzyme such as transketolase decreased appreciably above ethanol concentrations of 60 gL-1. It has been estimated that for ac­tively growing cells, as much as 30-50% of the cellular protein is comprised of ED enzymes [93]. However, the greatest difficulty for the commercial pro­duction of such enzymes is the low cell yield of Z. mobilis which is typically 0.02-0.03 gg-1 substrate sugar, compared to cell yields close to 0.5 gg-1 for many aerobically grown microorganisms.

4.2

Since the selection of spontaneous mutations during metabolic evolution has been a major component of the development of ethanologenic E. coli, many of the underlying changes contributing to ethanologenesis remain unidentified. Identification of these changes will aid in the development of biocatalysts with desired properties for production of other products.

3.1

Physiological Differences Conferring Ethanol Resistance to LY01

LY01 is a derivative of KO11 that was selected in rich medium for increased ethanol tolerance and yield [18]. As described above, LY01 had greater than 80% survival from brief exposure to 100 gL-1 ethanol, compared to only 10% survival for KO11 [18]. The transcriptomes of these two strains were compared in LB with glucose or xylose and with 0, 10, or 20 gL-1 ethanol [72]. Some 205 genes were differentially expressed in LY01 rela­tive to KO11, as determined by the student’s f-test; 49 of these genes were greater than twofold different in each comparison. Functional groups related to amino acid biosynthesis, cell processes, cell structure, central intermediary metabolism, and energy metabolism contained a high percentage of differ­entially expressed genes. Additionally, many stress-related genes, including those related to acid and osmotic stress, were differentially expressed.

Three major physiological differences between LY01 and KO11 were sug­gested by transcriptome data and supported by further analysis: increased glycine degradation, increased expression of genes related to betaine syn­thesis and uptake of protective osmolytes, and lack of FNR regulatory func­tion [72]. Normally, FNR regulates the expression of genes required for fer­mentation and anaerobic respiration (reviewed in [73]). Glycine metabolism and expression of FNR-regulated genes both impact the availability and dis­tribution of pyruvate. It is interesting to note that betaine synthesis genes are affected by FNR via ArcA [74,75]. Thus, the increased ethanol tolerance of LY01 seems to be a combination of several physiological factors, particularly those related to pyruvate partitioning and osmotic protection.

4

Lignin is separated out after glucose fermentation in the Maxifuel concept. Using a filter-type separator, it is possible to obtain the high dry weight lignin necessary to avoid simultaneous removal of xylose and ethanol still present in the liquid phase after initial hydrolysis and fermentation.

4.4

Fermentation

Biomass or agricultural residues consist of the polymers cellulose, hemicel — lulose, pectin, protein, and lignin. Of the carbohydrate monomers, xylose is second-most abundant after glucose in most plant cell walls [21]. Because the raw material cost is > 50% of the overall cost of the ethanol process, fermen­tation of xylose is needed to improve the yield and lower the production cost of ethanol since many biomasses and agricultural wastes contain xylose, in the order of 10-40% of the total carbohydrate mass. Fermentation of both xy­lose and glucose is therefore crucial to reduce the costs of ethanol production from lignocellulosic raw materials.

The baker’s yeast Saccharomyces cerevisiae is a desired process organism for fuel ethanol production due to its extensive use in current large-scale industrial ethanol production processes. Also, the excellent ethanol produc­tivity and tolerance towards ethanol and the inhibitors found in biomass hydrolysates are important reasons for using this organism, even though its natural xylose utilization capability is poor [22].

In the Maxifuel concept, a pentose and hexose fermenting thermophilic microorganism Thermoanaerobacter BG1 is used to ferment the residual sugars in the hydrolysate left after yeast fermentation [23]. Similar to the in­dustrial yeast strains, the thermophilic microorganism is able to grow under the harsh conditions provided by the hydrolysate whilst fermenting sug­ars efficiently. This genetically modified strain has been shown to produce

38.7 g/L or 5.4% v/v of ethanol in a continuous system running directly with non-detoxified lignocellulosic hydrolysate material. The yield from the process is 0.40 g/g total influent sugar or 78% of the theoretical possible value, and productivity is 0.85 g/L/h. The strain is tolerant to 7% of ethanol and higher dry weight in the pretreatment could be used for reaching this concentration.

Furthermore, it grows in temperatures of up to 75 °C, which eases the dis­tillation of ethanol from the reactor. Operation of the fermentation process at thermophilic conditions counteracts contamination by other bacteria, which is generally a problem for mesophilic yeast fermentation. During the residual sugar fermentation, between 0.5 and 1.1 mol of hydrogen/mol of substrate is produced. This is in the same magnitude as hydrogen yields from ded­icated dark fermentation of complex substrates such as sugar beet extract (1.0-1.7 mol hydrogen/mol substrate) [24] and molasses (0.52-1.58 mol hy­drogen/mol substrate) [8]. BG1 and all its mutants are covered by different patent applications.

To optimize the feasibility of the bioethanol production process the ther­mophilic fermentation is conducted in an immobilized reactor system. The immobilization of the fermenting organism in an up-flow reactor brings an array of important traits to the fermentation process like increased ethanol tolerance, high substrate conversions, and decreased sensitivity towards pro­cess imbalances.

4.5

E. coli has the capability of utilizing many different sugar substrates and produce a wide spectrum of fermentation products (Fig. 4). However, redirec­tion of a microorganism’s metabolism for the efficient production of a single compound is often far more complex than anticipated. The expression level of multiple genes, which may not be predictable, must be optimized for performance. Our success in generating microbial biocatalysts capable of pro-

ducing high titers of chemicals has been dependent on an approach that utilizes the organism’s natural ability to evolve. Genetically engineered mi­croorganisms require a period of time to adapt to the growth environment. This was accomplished by growing the microbial biocatalysts in the desired mineral salts medium with high sugar concentrations and allowing them to evolve in the new environment. This method has resulted in microbial bio­catalysts proficient in production of ethanol and other commodity products, demonstrating that this approach can be applied to many different microbial biocatalysts to improve the overall efficiency and titer.