Design of a New Multienzyme Complex Synthesis System Based on

Subscriber access provided by University of Sunderland

Article

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 ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b04401 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on November 1, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

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 Yang1†, Fei Li1†, Yangge Qiao1, Qinghua Zhou1, Zhiming Hu1, Yaojia He1, Yunjun Yan1, Li Xu1, Catherine Madzak2, Jinyong Yan*1, 3

Correspondence author: Jinyong Yan Correspondence author e-mail address: [email protected]

Affiliations and mailing addresses: 1Key

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 2GMPA,

AgroParisTech, INRA, Université Paris-Saclay, 78850, Thiverval-Grignon,

France 3Shenzhen

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

†Equal

contribution

ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract In order to address the challenges including 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 physico-chemical means, allowing to construct cell-associated two- or threeenzymes 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 17fold enhancement in 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 one pot fermentation process, integrate surface display of scaffoldin, extracellular production of dockerin-fused individual enzyme


Page 2 of 36

Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


components and highly efficient reassembly of the functional elements, through a genetically programmable process. Interestingly, the growing cells of MECs system provides a unique promotion effect to drive the substrate flux towards final product, through consuming of the by-product (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



Page 4 of 36

Introduction Considering the continuously growing demand on energy and resources, as well as everincreasing 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 biocatalysis1,2. 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 products3-7. However, the in vivo complex crosstalk of competitive metabolic pathways and target flux, together with the imbalance of enzymatic activities and co-factors involved in a given pathway, complicate in vivo microbial routes and limit their applications8,9. A cascade is refers to the combination of more than two reaction steps in a single reaction

pot

without

intermediate

separation1.

One-pot

multistep

cascade

biotransformations, catalyzed in vitro by multiple enzyme mixtures with defined composition, offer the advantage of controllable conditions10,11. Therefore, multiple enzymes-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 by-product formation and the fact that they require no biosynthesis of biomass12-15. Conventional cocktail mixtures of free enzymes allow to easily adjust the enzyme components ratio, but greatly suffer from expensive biocatalyst cost due to tedious individual enzymes preparation and poor recyclability16. Traditional


Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


combination of immobilized individual enzymes, supported on different materials, may enhance biocatalyst volumetric activity, stability, activity, selectivity or specificity, reusability to some extent but also introduces severe substrate/intermediate mass transfer limitation, leading to low reaction rate and conversion yield in multienzyme cascade17-19. Physico-chemical 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 purpose20,21. However, the method separately prepared individual enzymes and subsequently co-immobilizing them. Genetically engineered methods integrating enzyme preparation and co-immobilization are more attractive. In order to address the above mentioned issues, the natural self-assembly system of the cellulosome can be proposed as an alternative22. 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 biocatalysis23,24. This assembly strategy was already successfully applied in Saccharomyces cerevisiae for ethanol production from cellulosic substrates28. It also allowed the assembly, in Escherichia coli, of a three-enzyme complex, as a synthetic metabolon for fructose derivatives production29. However, the significant issues including relative ratio and relative position among these individual enzymes greatly affecting 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 dockerinenzyme 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 hyper-glycosylation 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 proteins29. 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, user-friendly, proportional and positional self-assembly system is urgently needed. Especially, unlike previous physico-chemical control of individual enzymes ratio and position32-35, 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, microorganisms, (especially microalgae) are widespread and renewable resources for biofuel and chemical production. The Thermomyces lanuginosus lipase (Tll), carboxylic acid reductase (CAR), aldehyde decarbonylase (ADC) were versatile enzymes as biocatalysts in biofuel and chemical production36-38. 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


Page 6 of 36

Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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, 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 compelling39. More interestingly, the broad scope of carbon source spectrum provides a unique possibility of promotion effect to drive the substrate flux towards final product in growing cells, through consuming of the by-products (such as glycerol) formed during cascade reactions40. In this context, the lipase (Tll) responsible for hydrolysis of TAGs to FFAs, and decarboxylase (OleTJE) responsible for decarboxylation of FFAs to alkenes were generated as MECs and used in 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 by-product, 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 threeenzyme 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.



Materials and methods Strains, plasmids and regents The Y. lipolytica strain Po1f and the plasmids used (pINA1317, pINA1297, pINA1317CWP110 and pQQR2) have been described previously41,42. Selection markers (ura3d1 and ura3d4, respectively non-defective and defective alleles41) present in the first three plasmids were flanked by loxR and loxP site sequences to generate corresponding modified plasmids pINA1317m, pINA1297m, pINA1317CWP110m, respectively. Plasmid pQQR2 carries and expresses 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 again42. 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, C3). pINA1317m and pINA1297m were used for expression and extracellular secretion of individual dockerin-containing fusion enzymes (Tll-D1, OleTJE-D2, CAR-D2, ADC-D3). pQQR2 was applied to rescue ura3d1 and ura3d4 selection markers. The genes of scaffold proteins CipA and DocA from 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 (C1-D1), CipC-celCCA (C2-D2) and


Page 8 of 36

Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 pRSFDuet-oletJE16. Various standard substrates were purchased from Sigma-Aldrich. All molecular genetic manipulation reagents and kits were brought from Beijing Solarbio Science & Technology Co., ltd (China).

Fusion protein construct, expression The DocA, celCCA and EngE dockerin-encoding genes, docA, celCCA and engE, were fused to individual enzyme genes tll, oletJE, car, adc by (G4S)2 linkers, using overlap extension PCR technology. Subsequently, the resulting fusions tll-docA (tll-D1), oletJEcelCCA (oletJE-D2), car-celCCA (car-D2), adc-engE (adc-D3) were inserted into pINA1317m and pINA1297m to create recombinant plasmids pINA1317m-tll-D1, pINA1297m-oletJE-D2, pINA1297m-car-D2, 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-C1-C2 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, 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 Po1f 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-Tll-D1, followed by screening on YNBD for Leu+ positive transformants, the ura3d1 selective marker integrated in the genome of Y. lipolytica-Tll-D1 was excised by Cre-mediated recombination to generate Ura- strain Y. lipolytica-Tll-D1. The replicative pQQR2 plasmid was lost after several generations33. The resulting Ura- strain Y. lipolytica-Tll-D1 was transformed by the recombinant plasmid pINA1297m-oletJE-D2, and a Ura+ positive transformant Y. lipolytica-(Tll-D1)- (OleTJE-D2) was selected after screening on YNBD plate using ura3d4 as 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-OleTJE-D2-C2, producing only one enzyme, the strain Y.lipolytica-Tll-OleTJE, with only extracellular co-expression 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-(Tll-D1)-(CAR-D2)-(ADC -D3)-C1-C2-C3, harboring


Page 10 of 36

Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 medium-containing flask and cultivated for 24 h at 30℃, 250 rpm, for pre-culture preparation. Cultivation of each strain was typically conducted by inoculation of 3 ml of pre-culture into a 500 ml flask containing 100 ml of YPD medium at 30℃, 250 rpm, for 48 h. During flask cultivation, the extracellularly secreted fusion components of enzyme-dockerins (such as Tll-D1, OleTJE-D2) were selfattached to respective cohesins (C1, C2) displayed on the cell surface, to form growing cells of MECs, called Y. lipolytica-(Tll-D1-C1)-(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-D1-C1) and Y. lipolytica-(OleTJE-D2-C2) were similarly prepared as control strains. The growing and resting cells of MECs, with self-assembled three-enzyme cascade (Tll-D1, CAR-D2, 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 al43. The free fatty acid (FFA) decarboxylation activity of OleTJE was measured according to a previously established method16. The FFA reduction activity of CAR, aldehyde



decarbonylation activity of ADC were detected according to a previously reported method38. The TAGs, FFAs, alkenes, alkanes were quantified by GC-MS analysis, according to a previous method established by Yan et al16.

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.

The conversion of TAG (microalgal oils) to fatty alkenes, 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-(TllD1-C1)-(OleTJE-D2-C2) or their counterparts as biocatalysts. These reactions were set, for different purposes, as follows: 0.625 mM-5 mM TAGs, 0.01%-0.5% H2O2, 10 mM TrisHCl buffer (pH 6.5). For growing cell biotransformation for alkene production, TAGs were added to the flask containing growing cells of MECs, at a post-culture time of 48 h, to initiate conversion. Cell-associated enzymatic activities, initial reaction rate, productivity and conversion yield were detected during growing cell conversion process.


Page 12 of 36

Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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 total contributions.

Results and discussion Assembly confirmation During cultivation of Y. lipolytica-(Tll-D1-C1)-(OleTJE-D2-C2) in 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 extracellular medium. Later, the two enzymes were gradually increasing 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, 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 culture supernatant and used as desirable resting cells of MECs. Our results demonstrated that fusions of the two dockerins (D1, D2) to Tll and OleTJE did not greatly affect their enzymatic functions (data not shown). More importantly, the dockerin containing fusions (Tll-D1, OleTJE-D2) and cohesin containing fusion (C1-C2) also retained their original 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 Y. lipolytica-(Tll-D1)-(OleTJE-D2) strain continuously increased during cultivation time, due to the absence of cohesin module (Figure 1A). Also, no cell-associated 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 cellassociated activity was detected, due to the lack of dockerin-enzyme fusions, which was a further confirmation of the anchoring and non-catalytic properties of C1-C2 (data not


Page 14 of 36

Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


shown). All these assembly validation results clearly demonstrate the simplicity and efficiency of the specific interaction between dockerins (D1, 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.

Figure 1. (A) Time course of free and assembled Tll and OleTJE activities during cultivation of MECs-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 yeast cell surface to form MECs during cultivation of strain Y. lipolytica-(Tll-D1-C1)-



(OleTJE-D2-C2).

Figure 2. A series of MECs with varied individual enzyme (Tll, OleTJE) proportional and positional effect, 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, (D) OleTJE:Tll=3:1. Characterization of substrate channeling of MECs The MECs with 1:1 ratio of Tll to OleTJE, in the form of whole cells Y. lipolytica-(Tll-D1C1)-(OleTJE-D2-C2), were prepared by spontaneous assembly of Tll-D1, OleTJE-D2 and cellC1-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.lipolytica-OleTJE-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 markedly a 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 MECs proximity effect (Figure 3B). Thus, the juxtaposition of the two enzymes in MECs


Page 16 of 36

Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


facilitated the initial reaction rate and the productivity, as well as the overall conversion yield, within shorter time, due to increased local concentrations of substrate, intermediate and enzymes. In contrast, mixtures of individual cell-bound enzymes (Y.lipolytica-Tll-D1C1, Y.lipolytica-OleTJE-D2-C2) and free enzymes both suffered from great diffusion effects, thus weakening the intermediate flux. Other MECs with different ratio 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, enzymes and cell surface, thus forming an efficient enzyme-substrate-cell complex for improving biocatalytic performances.

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



The 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 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)-(OleTJED2-C2)-(OleTJE-D2-C2)-(OleTJE-D2-C2), Y. lipolytica-

Figure 4. Individual enzyme proportional and positional effects of MECs on alkene


Page 18 of 36

Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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-OleTJEOleTJE-OleTJE (ratio 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. (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-D2C2)-(OleTJE-D2-C2)-(Tll-D1-C1) gave 67.2%, 70%, 78% and 84.4% conversion 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-D1-C1)-(OleTJE-D2-C2)-(OleTJE-D2-C2)- (OleTJE-D2-C2) and, to a lesser extent, Y. lipolytica-(OleTJE-D2-C2)-(Tll-D1-C1)- (OleTJE-D2-C2)-(OleTJE-D2-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(OleTJE-D2-C2)-(OleTJE-D2-C2)-(Tll- D1-C1)-(OleTJE-D2-C2) and Y. lipolytica-(OleTJE-D2C2)-(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 accomodate 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 cell-OleTJE-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 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 a by-product. We assumed that the yeast consumed by-product 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 decreased (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 by-product glycerol, but not FFAs, as a carbon source to support yeast further growth. To verify whether the yeast assimilates alkenes, a mixture with a defined composition of glucose, glycerol, FFAs, TAGs and alkenes, alkanes was tested for


Page 20 of 36

Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


yeast metabolization. The yeast preferentially utilized glucose or glycerol in the presence of FFAs, TAGs, alkenes, alkanes. Based on quantification of various components, we found that the yeast did not utilize TAGs, FFAs, alkenes, 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 by-product 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 by-product glycerol.

Figure 5. Conversion yield (A) and productivity (B) comparison between growing cells and resting cells of MECs. Extension of the assembly strategy from two enzymes to three enzymes to construct non-natural 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 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.

Figure 6. (A) Comparison of conversion yield of TAGs to alkanesbetween assembled three-enzymes (Tll, CAR, ADC) cascade MECs and their free enzyme mixture counterpart. (B) Schematic representation of cell-(ADC)2-CAR-Tl1 (ratio 2:1:1) Considering the poor activity of ADC38, 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 dramatically higher reaction rate and conversion yield compared to free enzymes (Figure 6A). Also, the Y.lipolytica-(ADC-D3-C3)-(CARD2-C2)-(Tll-D1-C1) construct, with lipase far away from the cell surface, displayed enhanced reaction rate and conversion yield compared to the one Y.lipolytica-(Tll-D1-C1)(ADC-D3-C3)-(CAR-D2-C2) with lipase close to the cell surface (Figure 6A). Although the


Page 22 of 36

Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


lipase located between CAR and ADC in the construct Y.lipolytica-(CAR-D2-C2)-(Tll-D1C1)-(ADC-D3-C3)2 has also less steric hindrance for accepting TAGs, compared to Y.lipolytica-(Tll-D1-C1)-(CAR-D2-C2)- (ADC-D3-C3)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-D2-C2)-(Tll-D1-C1) (Figure 6B). Similarly to 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 enzyme-catalyzed FFA-related reactions, in the form of a multienzyme module, to constitute an efficient cascade platform for the synthesis of a series of fatty acidderived products, is feasible and advantageous. For multiple enzyme cascade for hydrocarbon production, the high concentration of the product of one enzyme has potential toxicity towards 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 towards 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 self-assembled MECs Low cost microalgal oils were further bio-transformed into fatty alkenes, alkanes by the



use of these growing self-assembled MECs: Y.lipolytica-(OleTJE-D2-C2)3-(Tll-D1- C1) and Y.lipolytica-(ADC-D3-C3)2-(CAR-D2-C2)-(Tll-D1-C1). The totals of fatty alkenes and alkanes were summed up to 84%, 71% yields, respectively (Figure 7A, B), much higher than the respective yields of 32%, 8% obtained using mixtures of free individual enzymes. All the required times for achieving the highest yields using these self-assembled MECs were dramatically reduced from 6 h, 24 h to 3 h, 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 alkenes44-47, we proposed and demonstrated two highly efficient lipase based assembled cascade pathways (Tll-OleTJE, Tll-CAR-ADC) for alkenes and alkanes bioproduction. Alternative to the procedure of separately preparing individual enzymes and subsequently co-immobilizing them by physico-chemical means, we develop a one-pot genetically-encoded self-assembly system allowing to simultaneously produce individual enzymes and generate co-immobilized multienzymes.


Page 24 of 36

Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Alkene, alkane production from microalgal oils using assembled two- and threeenzymes cascade growing cell-(OleTJE)3-Tll (A), cell-(ADC)2-CAR-Tll (B) compared to their free counterparts. 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 cell-associated MECs. Diverse customized MECs with 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 much higher reaction rate and conversion yield due to substrate channeling, optimal ratio and positional arrangement of individual enzymes and cell surface supporting. The growing state of MECs exhibited a unique ability to utilize by-



product glycerol during TAGs hydrolysis and to push the reaction flux toward target products. Taken together, coupling of lipase-mediated TAGs 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 controllable proportion and position of individual enzymes for advanced biocatalysis and biosynthesis.

Supporting Information Individual enzyme sequences, cohesin sequences, dockerin sequences, main primers for construction purpose.

Notes The authors declare no competing financial interest.

Acknowledgments This work was supported by funding from the National Natural Science Foundation of China (Grant Number: NSFC31570793), the Fundamental Research Funds for the Central Universities (Grant Number: 2016YXMS255), Wuhan Morning Light Plan of Youth Science and Technology (Grant Number: 2017050304010292), Shenzhen Huazhong University of Science and Technology Research Institute Project (Grant Number:


Page 26 of 36

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


JCYJ20170818153352628), and the Startup Fund for Talent Scholars of Huazhong University of Science and Technology.

References 1. Schrittwieser, J.H.; Velikogne, S.; Hall, M.; Kroutil, W. Artificial Biocatalytic Linear Cascades for Preparation of Organic Molecules. Chem. Rev. 2018, 118, 270-348, DOI 10.1021/acs.chemrev.7b00033. 2. Marella, E.R.; Holkenbrink, C.; Siewers, V.; Borodina, I. Engineering Microbial Fatty Acid Metabolism for Biofuels and Biochemicals. Curr. Opin. Biotech. 2017, 50, 39-46, DOI 10.1016/j.copbio.2017.10.002. 3. Peralta-Yahya, P.P.; Zhang, F.; Cardayre, S.B.; Keasling, J.D. Microbial Engineering for the Production of Advanced Biofuels. Nature 2012, 488, 320-328, DOI 10.1038/nature11478. 4. Schirmer, A.; Rude, M.A.; Li, X.; Popova, E.; Cardayre, SB. Microbial Biosynthesis of Alkanes. Science 2010, 329, 559-562, DOI 10.1126/science.1187936. 5. Steen, E.J.; Kang, Y.; Bokinsky, G.; Hu, Z.; Schirmer, A.; McClure, A.; Cardayre, S.B.; Keasling, J.D. Microbial Production of Fatty-Acid-Derived Fuels and Chemicals from Plant Biomass. Nature 2010, 463, 559-562, DOI 10.1038/nature08721. 6. Qiao, K.; Wasylenko, T.M.; Zhou, K.; Xu, P.; Stephanopoulos, G. Lipid Production in Yarrowia lipolytica is Maximized by Engineering Cytosolic Redox Metabolism. Nat. Biotechnol. 2017, 35, 173-177, DOI 10.1038/nbt.3763. Epub 2017 Jan 16.



7. Yan, JY.; Yan, YJ.; Madzak, C.; Han, BN. Harnessing the Biodiesel Producing Microbes: from Genetic Engineering of Lipase to Metabolic Engineering of Fatty Acid Biosynthetic Pathway. Crit. Rev. Biotechnol. 2017, 37, 26-36, DOI 10.3109/07388551. 2015.1104531. 8. Kerfeld, C.A.; Heinhorst, S.; Cannon, G.C. Bacterial Microcompartments. Annu. Rev. Microbiol. 2010, 64, 391-408, DOI 10.1146/annurev.micro.112408.134211. 9. Good, M.C.; Zalatan, J.G.; Lim, W.A. Scaffold Proteins: Hubs for Controlling the Flow of Cellular Information. Science 2011, 332, 680-686, DOI 10.1126/science. 1198701. 10. Zhang, G.; Quin, M.B.; Schmidt-Dannert, C. Self-Assembling Protein Scaffold System for Easy in Vitro Coimmobilization of Biocatalytic Cascade Enzymes. ACS Catal. 2018, 8, 5611-5620, DOI 10.1021/acscatal.8b00986. 11. Zhang, YHP. Substrate Channeling and Enzyme Complexes for Biotechnological Applications. Biotechnol. Adv. 2011, 29, 715-725, DOI 10.1016/j.biotechadv.2011.05. 020. 12. Zhang, Y.; Hess H. Toward Rational Design of High-efficiency Enzyme Cascades. ACS Catal. 2017, 7, 6018-6027, DOI 10.1021/acscatal.7b01766. 13. Ross, J.F.; Bridges, A.; Fletcher, J.M.; Shoemark, D.; Alibhai, D.; Bray, H.E.V.; Beesley, J.L.; Dawson,W.M.; Hodgson, L.R.; Mantell, J.; Verkade, P.; Edge, C. M.; Sessions, R.B.; Tew, D.; Woolfson, D.N. Decorating Self-Assembled Peptide Cages with Proteins. ACS Nano 2017, 11, 7901-7914, DOI 10.1021/acsnano.7b02368. 14. Feng, J.; Mallapragada, S.K.; Narasimhan, B. Multienzyme Immobilization and Colocalization on Nanoparticles Enabled by DNA Hybridization. Ind. Eng. Chem. Res. 2015, 54, 10212-10220, DOI 10.1021/acs.iecr.5b01423}.


Page 28 of 36

Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15. Zhang, Y.; Ge, J.; Liu, Z. Enhanced Activity of Immobilized or Chemically Modified Enzymes. ACS Catal. 2015, 5, 4503-4513, DOI 10.1021/acscatal.5b00996. 16. Yan, J.; Liu, Y.; Wang, C.; Han, B.; Li S. Assembly of Lipase and P450 Fatty Acid Decarboxylase to Constitute a Novel Biosynthetic Pathway for Production of 1-alkene from Renewable Triglycerides and Oils. Biotechnol. Biofuels 2015, 8, 34-43, DOI 10.1186/s13068-015-0219-x. 17. Zhang, Y-HP. Production of Biocommodities and Bioelectricity by Cell-free Synthetic Enzymatic Pathway Biotransformations: Challenges and Opportunities. Biotechnol. Bioeng. 2010, 105, 663-677, DOI 10.1002/bit.22630. 18. Barbosa, O.; Torres, R.; Ortiz, C.; Berenguer-Murcia, A.; Rodrigues, R.C.; FernándezLafuente, R. Heterofunctional Supports in Enzyme Immobilization: from Traditional Immobilization

Protocols

to

Opportunities

in

Tuning

Enzyme

Properties.

Biomacromolecules 2013, 14, 2433-2462, DOI 10.1021/bm400762h. 19. Rodrigues, R.C.; Ortiz, C.; Berenguer-Murcia, A.; Torres, R.; Fernández-Lafuente, R. Modifying Enzyme Activity and Selectivity by Immobilization. Chem. Soc. Rev. 2013, 42, 6290-6307, DOI 10.1039/c2cs35231a. 20. Velasco-Lozano, S.; Benítez-Mateos, A.I.; López-Gallego, F. Co-immobilized Phosphorylated Cofactors and Enzymes as Self-Sufficient Heterogeneous Biocatalysts for Chemical Processes. Angew. Chem. Int. Edit. 2017, 56, 771-775, DOI 10.1002/ anie.201609758. 21. Peirce, S.; Virgen-Ortíz, J.J.; Tacias-Pascacio, V.G.; Rueda, N.; Bartolome- Cabrero,



Page 30 of 36

R.; Fernandez-Lopez, L.; Russo, M.E.; Marzocchella, A.; Fernandez- Lafuente, R. Development of Simple Protocols to Solve the Problems of Enzyme Coimmobilization. Application to Coimmobilize a Lipase and a β-galactosidase. RSC Adv. 2016, 6, 6170761715, DOI 10.1039/C6RA10906C. 22. Bae, J.; Morisaka, H.; Kuroda, K.; Ueda, M.Cellulosome Complexes: Natural Biocatalysts as Arming Microcompartments of Enzymes. J. Mol. Microbiol Biotechnol. 2013, 23, 370-378, DOI 10.1159/000351358. 23. Hyeon, J.E.; Shin, S.K.; Han, S.O. Design of Nanoscale Enzyme Complexes Based on Various Scaffolding Materials for Biomass Conversion and Immobilization. Biotechnol. J. 2016, 11, 1386-1396, DOI 10.1002/biot.201600039. 24. Lin, J.L.; Palomec, L.; Wheeldon, I. Design and Analysis of Enhanced Catalysis in Scaffolded Multienzyme Cascade Reactions. ACS Catal. 2014, 4, 505-511, DOI 10.1021/cs401009z. 25. Bayer, E. A.; Morag, E.; Lamed, R. The Cellulosome-a Treasure-Trove for Biotechnology.

Trends

Biotechnol.

1994,

12,

379-386,

DOI

10.1016/0167-

7799(94)90039-6. 26. Szczupak, A.; Aizik, D.; Moraïs, S.; Vazana, Y.; Barak, Y.; Bayer, EA.; Alfonta, L. The Electrosome: a Surface-Displayed Enzymatic Cascade in a Biofuel Cell's Anode and a High-Density Surface-Displayed Biocathodic Enzyme. Nanomaterials 2017, 7, 153, DOI 10.3390/nano7070153. 27. Stern, J.;

Moraïs, S.;

Ben-David, Y.;

Salama, R.;


Shamshoum, M.; Lamed, R.;

Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Shoham, Y.; Bayer, E.A.;

Mizrahib I. Assembly of Synthetic Functional Cellulosomal

Structures onto the Cell Surface of Lactobacillus plantarum, a Potent Member of the Gut Microbiome. Appl. Environ. Microbiol. 2018, 84, e00282-18, DOI 10.1128/AEM.0028218. 28. Fan, L.H.; Zhang, Z.J.; Yu, X.Y.; Xue, Y.X.; Tan, T.W. Self-Surface Assembly of Cellulosomes with Two Miniscaffoldins on Saccharomyces cerevisiae for Cellulosic Ethanol Production. Proc. Natl. Acad. Sci., USA. 2012, 109, 13260-13265, DOI 10.1073/pnas.1209856109. 29. You, C.; Zhang, YHP. Self-Assembly of Synthetic Metabolons through Synthetic Protein Scaffolds: One-Step Purification, Co-immobilization, and Substrate Channeling. ACS Synth. Biol. 2013, 2, 102-110, DOI 10.1021/sb300068g. 30. Sun, J., Wen, F., Si, T., Xu, J.H., Zhao, H.M. Direct Conversion of Xylan to Ethanol by Recombinant Saccharomyces cerevisiae Strains Displaying an Engineered Minihemicellulosome. Appl. Environ. Microbiol. 2012, 78, 3837-3845, DOI 10.1128/ AEM.07679-11. 31. Kim, H.J.; Kim, H.J. Yeast as An Expression System for Producing Virus-Like Particles: What Factors Do We Need to Consider? Lett. Appl. Microbiol. 2016, 64, 111123, DOI 10.1111/lam.12695. 32. Vriezema, D.M.; Garcia, P.M.L.; Oltra, N.S.; Hatzakis, N.S.; Kuiper, S.M.; Nolte, R.J.M.; Rowan, A.E.; Hest, J.C.M. Positional Assembly of Enzymes in Polymersome Nanoreactors for Cascade Reactions. Angew. Chem. Int. Ed. 2007, 46, 7378-7382, DOI



10.1002/anie.200701125. 33. Ai, H.; Jones, S.A.; Lvov, Y.M. Biomedical Applications of Electrostatic Layer-byLayer Nano-Assembly of Polymers, Enzymes, and Nanoparticles. Cell Biochem. Biophys. 2003, 39, 23-44, DOI 10.1385/CBB:39:1:23. 34. Ji, X.; Su, Z.; Wang, P.; Ma, G.; Zhang, S. Polyelectrolyte Doped Hollow Nanofibers for Positional Assembly of Bienzyme System for Cascade Reaction at O/W Interface. ACS Catal. 2014, 4, 4548-4559, DOI 10.1021/cs501383j. 35. Schoffelen, S.; Hest, J.C.M. Chemical Approaches for the Construction of MultiEnzyme Reaction Systems. Curr. Opin. Struc. Biol. 2013, 23, 613-621, DOI 10.1016/j.sbi.2013.06.010. 36. Fernández-Lafuente, R. Lipase from Thermomyces lanuginosus: Uses and Prospects as an Industrial Biocatalyst. J. Mol. Catal. B Enzym. 2010, 62, 197-212, DOI 10.1016/j.molcatb.2009.11.010. 37. Rude, M.A.; Baron, T.S.; Brubaker, S.; Alibhai, M.; Del Cardayre, S.B.; Schirmer, A. Terminal Olefin (1-alkene) Biosynthesis by a Novel p450 Fatty Acid Decarboxylase from Jeotgalicoccus Species. Appl. Environ. Microbiol. 2011, 77, 1718-1727, DOI 10.1128/AEM.02580-10. 38. Akhtar, M.K.; Turner, N.J.; Jones, P.R. Carboxylic Acid Reductase Is a Versatile Enzyme for the Conversion of Fatty Acids into Fuels and Chemical Commodities. Proc. Natl. Acad. Sci., USA. 2013, 110, 87-92, DOI 10.1073/pnas.1216516110. 39. Yan, J.; Han, B.; Gui, X.; Wang, G.; Xu, L.; Yan, Y. Madzak, C.; Pan, D.; Wang, Y.;


Page 32 of 36

Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Zha, G.; Jiao, L. Engineering Yarrowia lipolytica to Simultaneously Produce Lipase and Single Cell Protein from Agro-Industrial Wastes for Feed. Sci.Rep. 2018, 8, 758-767, DOI 10.1038/s41598-018-19238-9. 40. Qiao, Y.; Yang, K.; Zhou, Q.; Xu, Z.; Yan, Y.; Xu, L.; Madzak, C.; Yan, J. Engineering Yarrowia lipolytica for Sustainable Production of Fatty Acid Methyl Esters Using in Situ Self-Cycled Glycerol as a Carbon Source. ACS Sustain. Chem. Eng. 2018, 6, 7645-7651, DOI 10.1021/acssuschemeng.8b00492. 41. Madzak, C. Yarrowia lipolytica: Recent Achievements in Heterologous Protein Expression and Pathway Engineering. Appl. Microbiol. Biotechnol. 2015, 99, 4559-4577, DOI 10.1007/s00253-015-6624-z. 42. Fickers, P.; Dall, M.T.L.; Gaillardin, C.; Thonart, P.; Nicaud, J.M. New Disruption Cassettes for Rapid Gene Disruption and Marker Rescue in the Yeast Yarrowia lipolytica. J. Microbiol. Meth. 2003, 55, 727-737, DOI 10.1016/j.mimet.2003.07.003. 43. Yan, J.; Li, L.; Tang, Q.; Jiang, M.; Jiang, S. Preparation of A Crosslinked Bioimprinted Lipase for Enrichment of Polyunsaturated Fatty Acids from Fish Processing Waste. Appl. Biochem. Biotechnol. 2010, 162, 757-65, DOI 10.1007/ s12010-010-8910-7. 44. Zhu, Z.; Zhou, Y.J.; Kang, M.K.; Krivoruchko, A.; Buijs, N.A.; Nielsen, J. Enabling the Synthesis of Medium Chain Alkanes and 1-alkenes in Yeast. Metab. Eng. 2017, 44, 8188, DOI 10.1016/j.ymben.2017.09.007. 45. Zhou, Y.J.; Buijs, N.A.; Zhu, Z.; Gómez, D.O.; Boonsombuti, A.; Siewers, V.; Nielsen, J. Harnessing Yeast Peroxisomes for Biosynthesis of Fatty-acid-derived Biofuels and Chemicals with Relieved Side-pathway Competition. J. Am. Chem. Soc. 2016, 138, 1536815377, DOI 10.1021/jacs.6b07394.



46. Chen, B.; Lee, D.Y.; Chang, M.W. Combinatorial Metabolic Engineering of Saccharomyces cerevisiae for Terminal Alkene Production. Metab. Eng. 2015, 31, 53-61, DOI 10.1016/j.ymben.2015.06.009. 47. Herman, N.A.; Zhang, W. Enzymes for Fatty Acid-based Hydrocarbon Biosynthesis. Curr. Opin. Chem. Biol. 2016, 35, 22-28, DOI 10.1016/j.cbpa.2016.08. 009.


Page 34 of 36

Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Other authors and their e-mail addresses: Kaixin Yang1, [email protected] Fei Li1, [email protected] Yangge Qiao1, [email protected] Qinghua Zhou1, [email protected] Zhiming Hu1, [email protected] Yaojia He1, [email protected] Yunjun Yan1, [email protected] Li Xu1, [email protected] Catherine Madzak2, [email protected]



Graphical Abstract

Multienzymes were assembled to non-natural cascade with controllable proportion and position of individual enzymes for sustainable production of hydrocarbons.


Page 36 of 36

Design of a New Multienzyme Complex Synthesis System Based on

Recommend Documents