Design of a New Multienzyme Complex Synthesis System Based on

Oct 29, 2018 - In order to address the challenges of relative ratio and relative position among individual enzymes and substrate channeling in multien...
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Research Article Cite This: ACS Sustainable Chem. Eng. 2018, 6, 17035−17043

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Design of a New Multienzyme Complex Synthesis System Based on Yarrowia lipolytica Simultaneously Secreted and Surface Displayed Fusion Proteins for Sustainable Production of Fatty Acid-Derived Hydrocarbons Kaixin Yang,†,∥ Fei Li,†,∥ Yangge Qiao,† Qinghua Zhou,† Zhiming Hu,† Yaojia He,† Yunjun Yan,† Li Xu,† Catherine Madzak,‡ and Jinyong Yan*,†,§ Downloaded via UNIV OF OTAGO on December 17, 2018 at 08:21:13 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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Key Lab of Molecular Biophysics of Ministry of Education, Department of Biotechnology, College of Life Science and Technology, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, China ‡ GMPA, AgroParisTech, INRA, Université Paris-Saclay, 78850 Thiverval-Grignon, France § Shenzhen Huazhong University of Science and Technology Research Institute, Shenzhen Production and Research Base Block B, No. 9, Avenue 3 Yuexing, Yuehai Street, Nanshan District, Shenzhen 518057, China S Supporting Information *

ABSTRACT: In order to address the challenges of relative ratio and relative position among individual enzymes and substrate channeling in multienzyme biocatalysis, we propose a new one-pot genetically encoded self-assembly system as an alternative to the heavy procedure of separately preparing individual enzymes and subsequently co-immobilizing them by physicochemical means, allowing us to construct cell-associated two- or three-enzyme complexes, through the highly specific interaction of cohesin and dockerin domains. Our method for multienzyme complexes (MECs) assembled on Yarrowia lipolytica yeast cells is based on simultaneous surface display of a synthetic multiple cohesins backbone (scaffoldin) and extracellular secretion of dockerin-fusions of individual enzymes. This methodology was applied to fatty acid-derived hydrocarbon (alkenes, alkanes) production. The genetically tailored cell-associated MECs exhibited different proportional and positional effects through genetically encoded customization of the scaffoldin arrangement by varying the copy number and orientation of cohesin domains. The genetically encoded specific positional arrangement of individual enzymes was able to endow MECs with an obvious substrate channeling effect, reflected by a more than 17-fold enhancement in the initial reaction rate. Optimization of the co-immobilized multienzyme complex (through proportional and positional control of cohesin domains) gave much higher conversion yields (71−84%) compared to a free enzymes cocktail (8−32%). The resulting engineered yeast strains, designed for the one-pot fermentation process, integrate surface display of scaffoldin, extracellular production of dockerin-fused individual enzyme components, and highly efficient reassembly of the functional elements through a genetically programmable process. Interestingly, the growing cells of MEC system provides a unique promotion effect to drive the substrate flux toward the final product, through consuming the byproduct (glycerol) as a carbon source during cascade reactions. This adopted technology provides an efficient platform for creating tailored multienzymes for advanced cascade biosynthesis. KEYWORDS: Biocatalysis, Hydrocarbon, Lipase, Multienzyme complex, Self-assembly

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INTRODUCTION Considering the continuously growing demand on energy and resources, as well as ever-increasing environmental concerns aroused by rapid worldwide development, many efforts aim to develop environmentally friendly platforms for green and sustainable manufacturing of bio-based fuels, chemicals, pharmaceuticals, and materials, via microbial biosynthesis and advanced biocatalysis.1,2 Microbial biosynthesis based on genetic engineering, metabolic engineering, systems biology, and synthetic biology technologies has already shown its potential in the production of a variety of target products.3−7 © 2018 American Chemical Society

However, the in vivo complex crosstalk of competitive metabolic pathways and target flux, together with the imbalance of enzymatic activities and cofactors involved in a given pathway, complicate in vivo microbial routes and limit their applications.8,9 A cascade refers to the combination of more than two reaction steps in a single reaction pot without intermediate Received: September 1, 2018 Revised: October 19, 2018 Published: October 29, 2018 17035

DOI: 10.1021/acssuschemeng.8b04401 ACS Sustainable Chem. Eng. 2018, 6, 17035−17043

Research Article

ACS Sustainable Chemistry & Engineering separation.1 One-pot multistep cascade biotransformations, catalyzed in vitro by multiple enzyme mixtures with defined composition, offer the advantage of controllable conditions.10,11 Therefore, multiple enzyme-mediated cascade reactions, for various target production, from renewable and cost-effective feedstocks, have received more and more attention due to their flexible reaction conditions, their lesser byproduct formation, and the fact that they require no biosynthesis of biomass.12−15 Conventional cocktail mixtures of free enzymes allow us to easily adjust the enzyme components ratio but greatly suffer from expensive biocatalyst cost due to tedious individual enzymes preparation and poor recyclability.16 Traditional combination of immobilized individual enzymes, supported on different materials, may enhance biocatalyst volumetric activity, stability, activity, selectivity or specificity, and reusability to some extent, but it also introduces severe substrate/intermediate mass transfer limitation, leading to a low reaction rate and conversion yield in multienzyme cascade.17−19 Physicochemical co-immobilization of enzyme components onto the same solid carrier improves substrate transfer between individual enzymes but involves technological complication. Co-immbilization of enzymes and their cofactors, as well as galactosidase and lipase onto agarose−polyethylenimine based carriers, provided a solution for this purpose.20,21 However, the method separately prepared individual enzymes and then subsequently co-immobilized them. Genetically engineered methods integrating enzyme preparation and co-immobilization are more attractive. In order to address the issues mentioned above, the natural self-assembly system of the cellulosome can be proposed as an alternative.22 Scaffold protein-based assembly of individual enzymes through noncovalent cohesin and dockerin high affinity interaction is emerging as a powerful strategy to generate multienzyme complexes (MECs) for specific cascade biocatalysis.23−27 This assembly strategy was already successfully applied in Saccharomyces cerevisiae for ethanol production from cellulosic substrates.28 It also allowed the assembly, in Escherichia coli, of a three-enzyme complex, as a synthetic metabolon for fructose derivatives production.29 However, the significant issues, which include the relative ratio and relative position among these individual enzymes which greatly affect multienzyme biotransformation efficiency, have not been addressed. In addition, these previously reported assembly systems made use of at least two engineered strains of E. coli, with one strain generating the cohesin module and (an)other strain(s) producing the dockerin−enzyme modules. The efficiency of these systems involving S. cerevisiae or E. coli is reduced, notably due to unsatisfactory abilities of heterologous protein extracellular production. Also, a low yield of functional enzymes is produced due to hyperglycosylation of heterologous proteins in S. cerevisiae.30,31 For E. coli, due to poor secretion ability, disruption of cells is required for accessing fused target proteins.29 Therefore, a sufficient amount of fused proteins is obtained only at the expense of intensive labor and cost, for both of these two systems. Therefore, a highly efficient, rapid, robust, userfriendly, proportional, and positional self-assembly system is urgently needed. Especially, unlike previous physicochemical control of individual enzymes ratio and position,32−35 genetic regulation of proportional and positional effects of individual enzymes constitutes a promising alternative approach. Fatty alkenes and fatty alkanes are regarded as important fuels and chemicals. Triacylglycerols (TAGs) present in

various natural animals, plants, and microorganisms (especially microalgae) are widespread and renewable resources for biofuel and chemical production. The Thermomyces lanuginosus lipase (Tll), carboxylic acid reductase (CAR), and aldehyde decarbonylase (ADC) are versatile enzymes as biocatalysts in biofuel and chemical production.36−38 In the present study, instead of employing more than two recombinant strains, we choose Yarrowia lipolytica yeast as a chassis to develop an efficient one-pot genetically controlled proportional and positional assembly system, by simultaneously surface displaying a cohesin module and secreting dockerin−enzyme modules. Several previous concerns, including hyper-glycosylation, poor extracellular functional enzyme level, and need for cell disruption and for more than two strains employment will be all addressed. In addition, the exceptionally high cell density of Y. lipolytica yeast and its outstanding ability to utilize a broad range of low cost carbon sources are compelling.39 More interestingly, the broad scope of carbon source spectrum provides a unique possibility of a promotion effect to drive the substrate flux toward the final product in growing cells through consuming the byproducts (such as glycerol) formed during cascade reactions.40 In this context, the lipase (Tll) responsible for hydrolysis of TAGs to FFAs and the decarboxylase (OleTJE) responsible for decarboxylation of FFAs to alkenes were generated as MECs and used in the cascade reaction for conversion of TAGs to fatty alkenes in one pot. Enzymatic properties of the MECs, including activity, initial reaction rate, conversion yield, and conversion productivity, were comprehensively investigated. Proportional and positional effects of individual enzyme components present in MECs were optimized in a genetically encoded manner. Continual utilization of an in situ byproduct, glycerol, for increased alkene production was also verified in our growing cell system. Furthermore, the developed selfassembled two-enzyme MEC growing cell strategy was extended to constructing a three-enzyme MEC of Tll, carboxylic acid reductase (CAR) responsible for reduction of FFAs to fatty aldehydes, and aldehyde decarbonylase (ADC) responsible for decarboxylation of aldehydes to alkanes for efficient cascade conversion of TAGs to fatty alkanes in one pot.

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MATERIALS AND METHODS

Strains, Plasmids, and Regents. The Y. lipolytica strain Po1f and the plasmids used (pINA1317, pINA1297, pINA1317CWP110, and pQQR2) have been described previously.41,42 Selection markers (ura3d1 and ura3d4, respectively, nondefective and defective alleles41) present in the first three plasmids were flanked by loxR and loxP site sequences to generate corresponding modified plasmids pINA1317m, pINA1297m, and pINA1317CWP110m, respectively. Plasmid pQQR2 carries and expresses the Cre recombinase gene, which product recognizes loxR and loxP sites and triggers excision of the lox-flanked selection markers. Thus, the ura3d1 and ura3d4 selection markers already integrated in the genome of Y. lipolytica Po1f could be rescued and used for subsequent transformant screening again.42 E. coli DH 5α strain was employed for amplification of the various recombinant plasmids. Y. lipolytica Po1f was used as a host for expression of the various fused proteins, and all Y. lipolytica strains were cultivated in YPD medium supplemented with 10 mM CaCl2. pINA1317CWP110m was employed for surface display of cohesin modules (C1, C2, and C3). pINA1317m and pINA1297m were used for expression and extracellular secretion of individual dockerin-containing fusion enzymes (Tll-D1, OleTJE-D2, CAR-D2, and ADC-D3). pQQR2 was applied to rescue ura3d1 and ura3d4 selection markers. The genes of scaffold proteins CipA and DocA from 17036

DOI: 10.1021/acssuschemeng.8b04401 ACS Sustainable Chem. Eng. 2018, 6, 17035−17043

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ACS Sustainable Chemistry & Engineering

Figure 1. (A) Time course of free and assembled Tll and OleTJE activities during cultivation of MEC-producing strain Y. lipolytica-(Tll-D1-C1)(OleTJE-D2-C2) and control strain Y. lipolytica-(Tll-D1)-(OleTJE-D2) without cohesins. Extracellular Tll (OleTJE) activity of MECs refers to free extracellular individual enzymes during cultivation of Y. lipolytica-(Tll-D1-C1)-(OleTJE-D2-C2). Tll (OleTJE) activity of MECs refers to assembled cell-associated MECs during cultivation of Y. lipolytica-(Tll-D1-C1)-(OleTJE-D2-C2). Tll (OleTJE) activity of non-MECs refers to free extracellular individual enzymes during cultivation of control strain Y. lipolytica-(Tll-D1)-(OleTJE-D2). (B) Schematic representation of secreted Tll-D1 and OleTJE-D2 self-assembly onto C1−C2 displayed on the yeast cell surface to form MECs during cultivation of strain Y. lipolytica-(Tll-D1-C1)-(OleTJED2-C2). Clostridium acetobutylicum, CipC and celCCA from Clostridium cellulolyticum, and CbpA and EngE from Clostridium cellulovorans were used as three cohesin−dockerin pairs, namely, CipA-DocA (C1D1), CipC-celCCA (C2-D2), and CbpA-EngE (C3-D3). The genes encoding carboxylic acid reductase (CAR), aldehyde decarbonylase (ADC), and the above three pairs of cohesin−dockerin sequences, as well as all oligonucleotide primers, were synthesized by Tianyi Huiyuan Company (China). The genes Tll and OleTJE were subcloned from previous constructs pRSFDuet-tll and pRSFDuetoletJE.16 Various standard substrates were purchased from SigmaAldrich. All molecular genetic manipulation reagents and kits were brought from Beijing Solarbio Science & Technology Co., Ltd. (China). Fusion Protein Construct and Expression. The DocA, celCCA, and EngE dockerin-encoding genes, docA, celCCA, and engE, respectively, were fused to individual enzyme genes tll, oletJE, car, and adc by (G4S)2 linkers, using overlap extension PCR technology. Subsequently, the resulting fusions tll-docA (tll-D1), oletJE-celCCA (oletJE-D2), car-celCCA (car-D2), and adc-engE (adc-D3) were inserted into pINA1317m and pINA1297m to create recombinant plasmids pINA1317m-tll-D1, pINA1297m-oletJE-D2, pINA1297m-car-D2, and pINA1297m-adc-D3, respectively. Similarly, the corresponding cohesin-encoding genes CipA (C1), CipC (C2), and CbpA (C3) were fused using (G4S)2 linkers into pINA1317CWP110m to generate recombinant plasmids pINA1317CWP110m-C 1 -C 2 and pINA1317CWP110m-C1-C2-C3. In order to regulate the ratio of lipase Tll to OleTJE and their relative position, the copy number and position of cohesins could be genetically adjusted when constructing pINA1317CWP110m-cohesins. The specific interaction between the cohesin and dockerin pairs (C1-D1, C2-D2, and C3-D3) enabled assembly of a series of MECs in terms of varied ratios and positions. The recombinant plasmid pINA1317m-tll-D1 was used to transform competent cells of Y. lipolytica Po 1f using lithium acetate method and transformation mixture was plated on YNBD medium to screen Ura+ positive transformants Y. lipolytica-Tll-D1. Through transformation with pQQR2 harboring recombinase Cre of the host Y. lipolytica-TllD1, followed by screening on YNBD for Leu+ positive transformants, the ura3d1 selective marker integrated in the genome of Y. lipolyticaTll-D1 was excised by Cre-mediated recombination to generate Ura− strain Y. lipolytica-Tll-D1. The replicative pQQR2 plasmid was lost after several generations.33 The resulting Ura− strain Y. lipolytica-TllD1 was transformed by the recombinant plasmid pINA1297m-oletJED2, and a Ura+ positive transformant Y. lipolytica-(Tll-D1)-(OleTJED2) was selected after screening on YNBD plate using ura3d4 as a

selection marker. Similarly, Cre recombinase-mediated excision of ura3d4 marker from the genome of Y. lipolytica-(Tll-D1)-(OleTJE-D2) was repeated as described above. The recombinant plasmid pINA1317CWP110m-C1-C2 was used to transform Ura− Y. lipolytica-(Tll-D1)-(OleTJE-D2) host to obtain Ura+ transformant Y. lipolytica-(Tll-D1)-(OleTJE-D2)-C1-C2 on YNBD plate. For comparison, the control strains Y. lipolytica-Tll-D1-C1 and Y. lipolytica-OleTJED2-C2, producing only one enzyme, the strain Y. lipolytica-Tll-OleTJE, with only extracellular coexpression of both enzymes without scaffoldin C1−C2, and the strain Y. lipolytica-Tll-linker-OleTJE, with their conventionally fused expression without scaffoldin C1−C2, were also constructed. Similarly, the recombinant strains Y. lipolytica-(TllD1)-(CAR-D2)-(ADC-D3)-C1-C2-C3, harboring three cascade enzymes genes (tll-car-adc) and three cohesin genes (C1−C2−C3), were constructed, based on Cre recombinase-mediated rescuing of selection markers. A typical cultivation was performed as follows. A single clone of each engineered strain was inoculated into a 15 mL YPD mediumcontaining flask and cultivated for 24 h at 30 °C, at 250 rpm, for preculture preparation. Cultivation of each strain was typically conducted by inoculation of 3 mL of preculture into a 500 mL flask containing 100 mL of YPD medium at 30 °C, at 250 rpm, for 48 h. During flask cultivation, the extracellularly secreted fusion components of enzyme-dockerins (such as Tll-D1 and OleTJE-D2) were self-attached to respective cohesins (C1 and C2) displayed on the cell surface to form growing cells of MECs, called Y. lipolytica-(Tll-D1C1)-(OleTJE-D2-C2). The engineered Y. lipolytica-(Tll-D1-C1)(OleTJE-D2-C2) whole cells with assembled fused proteins were harvested by centrifugation and washed, to form resting cells of MECs. Also, single enzyme-bearing whole cells Y. lipolytica-(Tll-D1C1) and Y. lipolytica-(OleTJE-D2-C2) were similarly prepared as control strains. The growing and resting cells of MECs, with selfassembled three-enzyme cascade (Tll-D1, CAR-D2, and ADC-D3) and scaffoldin (C1−C2−C3), namely, Y. lipolytica-(Tll-D1)-(CAR-D2)(ADC-D3)-C1-C2-C3 were prepared in the same manner. Enzymatic Activity, Intermediate, and Product Assays. Lipase activity of Tll was determined based on the method described by Yan et al.43 The free fatty acid (FFA) decarboxylation activity of OleTJE was measured according to a previously established method.16 The FFA reduction activity of CAR and the aldehyde decarbonylation activity of ADC were detected according to a previously reported method.38 The TAGs, FFAs, alkenes, and alkanes were quantified by GC-MS analysis, according to a previous method established by Yan et al.16 17037

DOI: 10.1021/acssuschemeng.8b04401 ACS Sustainable Chem. Eng. 2018, 6, 17035−17043

Research Article

ACS Sustainable Chemistry & Engineering Characterization of Substrate Channeling of MECs. Using pure tripalmitin (TAG with three C16:0 fatty acyl chains) as substrate, based on Tll hydrolysis coupled to OleTJE decarboxylation, the conversion of TAGs to alkenes was set as a model reaction. Using the mixed free enzyme solution of individual enzymes (Tll and OleTJE) with the same composition as a reference, the catalytic performances of resting cells of assembled MECs were characterized in terms of initial reaction rate, productivity, and conversion yield. The substrate channeling was reflected as an enhancement of initial reaction rate. Conversion of TAG (Microalgal Oils) to Fatty Alkenes and Alkanes. For resting cell biotransformation for alkene production, Tll-OleTJE mediated tandem reactions from TAGs to alkenes were performed using resting cells of Y. lipolytica-(Tll-D1-C1)-(OleTJE-D2C2) or their counterparts as biocatalysts. These reactions were set, for different purposes, as follows: 0.625−5 mM TAGs, 0.01−0.5% H2O2, and 10 mM Tris-HCl buffer (pH 6.5). For growing cell biotransformation for alkene production, TAGs were added to the flask containing growing cells of MECs, at a postculture time of 48 h, to initiate conversion. Cell-associated enzymatic activities, initial reaction rate, productivity, and conversion yield were detected during the growing cell conversion process. For Y. lipolytica-(Tll-D1-C1)-(CAR-D2-C2)-(ADC-D3-C3) with assembled Tll, CAR, and ADC, the biotransformation of TAGs to fatty alkanes, using resting cells or growing cells of MECs, was similarly performed as described above. The resting cell and growing cell systems were supplemented with 5 mM TAGs or microalgal oils, 30 mM NADPH, 30 mM ATP, and 300 mM MgCl2. In the growing cell bioproduction system, Y. lipolytica secreted individual enzymes into extracellular medium and self-assembled individual enzymes were displayed on the cell surface as MECs. Therefore, the free extracellular solution of individual enzymes and the cell-associated MECs both made contributions to the accumulation of fatty acid-derived products. In order to distinguish between these two types of contributions, an engineered Y. lipolytica strain harboring only the corresponding dockerin−enzyme modules, without cohesin modules, was set as a control for the contribution of free extracellular individual enzymes to target product. Thus, the contributions originated from the MECs were obtained by subtracting the extracellular free enzyme contributions from the total contributions.

specific mutual binding abilities to accomplish self-assembly of enzyme components onto the cell surface. In contrast to the case of Y. lipolytica-(Tll-D1-C1)-(OleTJE-D2-C2), the extracellular enzymatic activities of the Y. lipolytica-(Tll-D1)-(OleTJED2) strain continuously increased during cultivation time, due to the absence of cohesin module (Figure 1A). Also, no cellassociated activity was detected for the resting cells of Y. lipolytica-(Tll-D1)-(OleTJE-D2), namely, without cohesin− dockerin-based self-assembly. For the other control strain Y. lipolytica-C1-C2, neither extracellular activity nor cell-associated activity was detected, due to the lack of dockerin−enzyme fusions, which was a further confirmation of the anchoring and noncatalytic properties of C1−C2 (data not shown). All these assembly validation results clearly demonstrate the simplicity and efficiency of the specific interaction between dockerins (D1 and D2) present in enzyme components and cohesins present in the synthetic scaffoldin (C1−C2). Especially, in order to achieve a broad range of catalytic performances as the two-step reaction required, a variety of MECs were generated through rational design of a series of scaffoldins with a diverse range of cohesin copy number and order (Figure 2). These variants were easily implemented by recombinant DNA technology when constructing scaffoldin fusions.

RESULTS AND DISCUSSION Assembly Confirmation. During cultivation of Y. lipolytica-(Tll-D1-C1)-(OleTJE-D2-C2) in a flask, the lipase (Tll) and fatty acid decarboxylase (OleTJE) activities in the extracellular medium varied with increasing time (Figure 1A). Since the nutritional energy supply was mainly in support of synthesizing yeast biomass during the first 18 h, very low activities of both enzymes were then detected in the extracellular medium. Later, the two enzymes were gradually increased to achieve the high activity levels of 168 U/mL for Tll and 6 μM for OleTJE after 36 h. The extracellular activities then decreased from the time point of 36 h due to large amounts of extracellular free dockerin-containing enzymes binding to synthetic scaffoldin C1−C2, and the surface displayed on the cell surface through dockerin-cohesin specific interactions. This assembly hypothesis was confirmed by measuring the assembled enzyme activities on harvested whole cells. Detection of the assembled enzyme activities revealed that harvested cells from 48 h of culture exhibited high activities (Figure 1A); they can thus be separated from the culture supernatant and can be used as desirable resting cells of MECs. Our results demonstrated that fusions of the two dockerins (D1 and D2) to Tll and OleTJE did not greatly affect their enzymatic functions (data not shown). More importantly, the dockerin-containing fusions (Tll-D1 and OleTJE-D2) and cohesin-containing fusion (C1−C2) also retained their original

Figure 2. A series of MECs with varied individual enzyme (Tll and OleTJE) proportional and positional effects, obtained through regulation of cohesin copy number and order when constructing scaffoldins via recombinant DNA technology. (A) Tll/OleTJE = 1:1; (B) Tll/OleTJE = 1:3; (C) OleTJE/Tll = 1:1; and (D) OleTJE/Tll = 3:1.

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Characterization of Substrate Channeling of MECs. The MECs with a 1:1 ratio of Tll to OleTJE, in the form of whole cells of Y. lipolytica-(Tll-D1-C1)-(OleTJE-D2-C2), were prepared by spontaneous assembly of Tll-D1, OleTJE-D2, and cell-C1-C2 during shaking cultivation of Y. lipolytica-(Tll-D1)(OleTJE-D2)-C1-C2 (Figure 1B, Figure 2A). The harvested resting cells of MECs were compared to the equivalent mixture of strains of equivalent Y. lipolytica-Tll-D1-C1 and Y. lipolyticaOleTJE-D2-C2, as well as to a solution mixture of equivalent free Tll and OleTJE, for the conversion of TAGs to alkenes. The 1:1 MECs exhibited a markedly more than 17-fold increase in initial reaction rate compared to the latter two mixtures (Figure 3A). Unlike the significant loss of intermediate FFAs to reaction medium, due to free diffusion, observed in free enzyme solution, the intermediate FFAs passed from the active site of Tll to the active site of OleTJE due to the MECs proximity effect (Figure 3B). Thus, the juxtaposition of the two enzymes in MECs facilitated the initial reaction rate and the productivity, as well as the overall conversion yield, within a shorter time, due to increased local 17038

DOI: 10.1021/acssuschemeng.8b04401 ACS Sustainable Chem. Eng. 2018, 6, 17035−17043

Research Article

ACS Sustainable Chemistry & Engineering

Figure 3. Experimental validation (A) and schematic representation (B) of substrate channeling for the resting cells of MECs with a 1:1 ratio of Tll to OleTJE. Substrate channeling was reflected as the initial reaction rate enhancement through comparison of assembled MECs with equivalent nonassembled mixture of whole cells or with free solution of individual enzymes.

Figure 4. Individual enzyme proportional and positional effects of MECs on alkene conversion yield. (A) Individual enzyme proportional effect on alkene conversion yield, under the fixed order of cell-Tll-OleTJE. (B) Schematic representation of cell-Tll-OleTJE-OleTJE-OleTJE (ratio of 1:3). (C) Individual enzyme positional effect on alkene conversion yield, under the fixed 1:3 ratio of Tll to OleTJE. (D) Schematic representation of cellOleTJE-OleTJE-OleTJE-Tll..

concentrations of substrate, intermediate, and enzymes. In contrast, mixtures of individual cell-bound enzymes (Y. lipolytica-Tll-D1-C1 and Y. lipolytica-OleTJE-D2-C2) and free enzymes both suffered from great diffusion effects, thus weakening the intermediate flux. Other MECs with different ratios of individual enzymes also showed substrate channeling effects, as indicated by increased initial reaction rates, productivities, and conversion yields (data not shown). The flexibility of the scaffold protein offers a structure basis for adequately, sufficiently and tightly binding among substrate, enzymess and cell surface, thus forming an efficient enzyme− substrate−cell complex for improving biocatalytic performances. Ratio and Positional Effects on Conversion of TAGs to Alkenes by Resting Cells of MECs. We fixed at first the

position order of individual enzymes as cell-Tll-OleTJE, in order to investigate the ratio effect (Figure 4B). As shown in Figure 4A, the highest conversion yield, of 67.2%, was achieved at a 1:3 ratio of Tll to OleTJE. Interestingly, different positional effects, on both the productivity and conversion yield, were observed by employing a panel of MECs of cell-Tll-OleTJE with varied positional arrangements of individual enzymes. As described in Figure 4C, under the fixed 1:3 ratio of Tll to OleTJE, the positional arrangement of Y. lipolytica-(Tll-D1-C1)-(OleTJE-D2-C2)(OleTJE-D2-C2)-(OleTJE-D2-C2), Y. lipolytica-(OleTJE-D2-C2)(Tll-D1-C1)-(OleTJE-D2-C2)-(OleTJE-D2-C2), Y. lipolytica(OleTJE-D2- C2)-(OleTJE-D2-C2)-(Tll-D1-C1)-(OleTJE-D2-C2), and Y. lipolytica-(OleTJE-D2-C2)-(OleTJE-D2-C2)-(OleTJE-D2C2)-(Tll-D1-C1) gave 67.2%, 70%, 78%, and 84.4% conversion 17039

DOI: 10.1021/acssuschemeng.8b04401 ACS Sustainable Chem. Eng. 2018, 6, 17035−17043

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ACS Sustainable Chemistry & Engineering

Figure 5. Conversion yield (A) and productivity (B) comparison between growing cells and resting cells of MECs.

Figure 6. (A) Comparison of conversion yield of TAGs to alkanes between assembled three-enzymes (Tll, CAR, ADC) cascade MECs and their free enzyme mixture counterpart. (B) Schematic representation of cell-(ADC)2-CAR-Tl1 (ratio of 2:1:1).

yields, respectively. If the Tll responsible for the hydrolysis of substrate TAGs with higher molecular weight was placed in close proximity to the cell surface, the resulting constructs Y. lipolytica-(Tll-D 1 -C 1 )-(OleT JE -D 2 -C 2 )-(OleT JE -D 2 -C 2 )(OleTJE-D2-C2) and, to a lesser extent, Y. lipolytica-(OleTJE-D2C 2 )-(Tll-D1 -C 1 )-(OleT JE -D 2-C 2 )-(OleT JE -D 2 -C2 ) showed lower productivities and conversion yields. In contrast, when the OleTJE responsible for decarboxylation of FFAs was in close proximity to the cell surface, as for Y. lipolytica-(OleTJED2-C2)-(OleTJE-D2-C2)-(Tll- D1-C1)-(OleTJE-D2-C2) and Y. lipolytica-(OleTJE-D2-C2)-(OleTJE-D2-C2)-(OleTJE-D2-C2)(Tll-D1-C1) (Figure 4D), higher productivities and conversion yields were achieved. The varied positional effects might be attributed to steric hindrance caused by different individual enzyme components’ positional arrangements. TAGs with three fatty acyl chains require a large space to be able to enter the lipase active site. The arrangements of the cell-Tll-OleTJE series of constructs present a crowded region between cell surface and adjacent lipase (Tll), thus the steric hindrance effect possibly hampers TAG connection to Tll. While in the case of cell-OleTJE-Tll series of constructs, there is more room to accommodate TAGs with higher molecule weight, due to lipase being farther away from the cell surface. In contrast, when OleTJE was closest to the cell surface, as in the cellOleTJE-Tll series of constructs, available space was probably sufficient for intermediate FFAs with lower molecular weight. Conversion of TAGs to Alkenes by Growing Cells of MECs. In situ bioproduction of alkenes using growing cells of MECs was investigated. To our surprise, growing cells of the MECs catalytic system reached a much higher yield within a much shorter time, compared to the resting cells obtained from

equivalent cultures (Figure 5). The hydrolysis of TAGs produced intermediate FFAs and glycerol as byproducts. We assumed that the yeast consumed byproduct glycerol to drive the reversible hydrolysis reaction, thus further favoring the following decarboxylation reaction. To verify this hypothesis, we employed growing cells of Y. lipolytica-Tll-D1-C1 in a hydrolysis reaction alone. As we expected, the glycerol formed during hydrolysis of TAGs was gradually consumed, but the FFA level did not decrease (data not shown). Simultaneously, the yeast biomass, together with the lipase activity, was continually increasing during the considered period of time, compared to growing cells without addition of TAGs. The results revealed that the yeast utilized byproduct glycerol, but not FFAs, as a carbon source to support further yeast growth. To verify whether the yeast assimilates alkenes, a mixture with a defined composition of glucose, glycerol, FFAs, TAGs, and alkenes, alkanes were tested for yeast metabolization. The yeast preferentially utilized glucose or glycerol in the presence of FFAs, TAGs, alkenes, and alkanes. On the basis of quantification of various components, we found that the yeast did not utilize TAGs, FFAs, alkenes, or alkanes before exhaustion of glucose and glycerol (data not shown). Therefore, the priority of glycerol over FFAs, TAGs, alkenes, and alkanes for assimilation by the yeast could be employed to lessen the inhibition of byproduct and to forward the overall reaction rate and conversion without consuming the substrate, intermediate, and product. This unique feature of the growing cell system could be extended to other advanced biocatalysis when forming byproduct glycerol. Extension of the Assembly Strategy from Two Enzymes to Three Enzymes to Construct Non-Natural 17040

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Figure 7. Alkene and alkane production from microalgal oils using assembled two- and three-enzyme cascade growing cell-(OleTJE)3-Tll (A) and cell-(ADC)2-CAR-Tll (B) compared to their free counterparts.

Biocatalytic Cascade for Alkane Production. Three enzymes, including lipase Tll, carboxylic acid reductase (CAR), and aldehyde decarbonylase (ADC), were assembled into MECs of Tll-CAR-ADC by the strategy described above. We generated the two constructs of Y. lipolytica-(Tll-D1-C1)(CAR-D2-C2)-(ADC-D3-C3) for conversion of TAGs to alkanes in resting cell systems. As described in Figure 6, the conversion yields of alkanes were dramatically improved in comparison to those of mixtures of free individual enzymes. The three-enzymes cascade MECs-mediated biotransformation significantly exceeded previous conversion efficiency for synthesis of alkanes. Considering the poor activity of ADC,38 a second copy of cohesin C3 was introduced into the scaffold construct: C1− C2−(C3)2. The resulting self-assembled Y. lipolytica-(Tll-D1C1)-(CAR-D2-C2)-(ADC-D3-C3)2 gave a dramatically higher reaction rate and conversion yield compared to free enzymes (Figure 6A). Also, the Y. lipolytica-(ADC-D3-C3)-(CAR-D2C2)-(Tll-D1-C1) construct, with lipase far away from the cell surface, displayed an enhanced reaction rate and conversion yield compared to the one Y. lipolytica-(Tll-D1-C1)-(ADC-D3C3)-(CAR-D2-C2) with lipase close to the cell surface (Figure 6A). Although the lipase located between CAR and ADC in the construct Y. lipolytica-(CAR-D2-C2)-(Tll-D1-C1)-(ADCD3-C3)2 has also less steric hindrance for accepting TAGs, compared to Y. lipolytica-(Tll-D1-C1)-(CAR-D2-C2)-(ADC-D3C3)2, the bidirectional channeling reduces the reaction flux toward end product, unlike the concentrated single directional substrate channeling by Y. lipolytica-(ADC-D3-C3)2-(CAR-D2C2)-(Tll-D1-C1) (Figure 6B). Similarly to the above alkene production using growing cells of MECs, the growing cells of MECs exhibited moderately higher conversion yields (81%) for alkane production than the resting cells of respective MECs (71%). Taken together, coupling of Tll mediated TAG hydrolysis to other enzymecatalyzed FFA-related reactions, in the form of a multienzyme module, to constitute an efficient cascade platform for the synthesis of a series of fatty acid-derived products is feasible and advantageous. For multiple enzyme cascade for hydrocarbon production, the high concentration of the product of one enzyme has potential toxicity toward the enzyme. Especially in the case of three-enzyme cascade for alkane production, the high concentration of the product (fatty aldehyde) of the second

enzyme (CAR) has potential toxicity toward the CAR. The property of spatial close to each enzyme in co-immobilized enzymes significantly reduces the potential toxicity and gives increased yields. Cascade Conversion of Renewable Natural Oils to Fatty Acid-Derived Hydrocarbons Using Growing SelfAssembled MECs. Low cost microalgal oils were further biotransformed into fatty alkenes and alkanes by the use of these growing self-assembled MECs: Y. lipolytica-(OleTJE-D2C2)3-(Tll-D1-C1) and Y. lipolytica-(ADC-D3-C3)2-(CAR-D2C2)-(Tll-D1-C1). The totals of fatty alkenes and alkanes were summed up to 84% and 71% yields, respectively (Figure 7A and B), much higher than the respective yields of 32% and 8% obtained using mixtures of free individual enzymes. All the required times for achieving the highest yields using these selfassembled MECs were dramatically reduced from 6 and 24 h to 3 and 8 h, respectively, compared with the corresponding mixture counterparts of free individual enzymes. As alternatives to in vivo metabolic engineering of S. cerevisiae for biosynthesis of alkanes and alkenes,44−47 we proposed and demonstrated two highly efficient lipase-based assembled cascade pathways (Tll-OleTJE and Tll-CAR-ADC) for alkenes and alkanes bioproduction. Alternative to the procedure of separately preparing individual enzymes and subsequently co-immobilizing them by physicochemical means, we develop a one-pot genetically encoded self-assembly system allowing us to simultaneously produce individual enzymes and generate co-immobilized multienzymes.

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CONCLUSIONS Through fusion of dockerin to individual enzymes and specific binding between cohesin and dockerin, Y. lipolytica engineered strains capable of simultaneous extracellular individual fatty acid-related enzymes secretion and surface display of tailored cohesins proved able to promote self-assembly of cellassociated MECs. Diverse customized MECs with a varied positional effect and ratio of individual enzymes could be generated by refining the constructs, using a genetically encoded programmable assembly manner. The assembled MECs exhibited multiple advantages compared to their free enzyme mixed counterparts, such as a much higher reaction rate and conversion yield due to substrate channeling, optimal ratio and positional arrangement of individual enzymes, and 17041

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cell surface support. The growing state of MECs exhibited a unique ability to utilize byproduct glycerol during TAG hydrolysis and to push the reaction flux toward target products. Taken together, the coupling of lipase-mediated TAG hydrolysis to other related enzyme-catalyzed FFA reactions can be modularized into a multienzyme complex based on simultaneous extracellular expression and surface display to constitute a highly efficient cascade platform for the synthesis of a broad range of fatty acid-derived products. This engineered Y. lipolytica system provides an efficient one-pot integrated assembly platform to generate MECs with a controllable proportion and position of individual enzymes for advanced biocatalysis and biosynthesis.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.8b04401. Individual enzyme sequences, cohesin sequences, dockerin sequences, and main primers for the construction purpose (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yunjun Yan: 0000-0002-4225-6261 Jinyong Yan: 0000-0003-2543-203X Author Contributions ∥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by funding from the National Natural Science Foundation of China (NSFC31570793), the Fundamental Research Funds for the Central Universities (2016YXMS255), the Wuhan Morning Light Plan of Youth Science and Technology ( 2017050304010292), the Fund from Science, Technology and Innovation Commission of Shenzhen Municipality (JCYJ20170818153352628), and the Startup Fund for Talent Scholars of Huazhong University of Science and Technology.

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