Development of Next Generation Synthetic Biology Tools for Use in

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Development of next generation synthetic biology tools for use in Streptomyces venezuelae Ryan M. Phelan, Daniel Sachs, Shayne J. Petkiewicz, Jesus F. Barajas, Jacquelyn M. Blake-Hedges, Mitchell G Thompson, Amanda Reider Apel, Blake J. Rasor, Leonard Katz, and Jay D Keasling ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00202 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 8, 2016

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ACS Synthetic Biology

Development of next generation synthetic biology tools for use in Streptomyces venezuelae 1,2

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1

1

Ryan M. Phelan, Daniel Sachs, Shayne J. Petkiewicz, Jesus F. Barajas, Jacquelyn M. Blake-Hedges, 5 1 3 2 1,2,6,7,# Mitchell G. Thompson, Amanda Reider Apel, Blake J. Rasor, Leonard Katz , Jay D. Keasling 1

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Joint Bioenergy Institute, 5885 Hollis Street, Emeryville, CA 94608; QB3 Institute, University of California, Berkeley, California 94270, United 3 States; Department of Biology, Miami University, 212 Pearson Hall, Oxford, Ohio 45046; Department of Chemistry, University of California, 5 6 Berkeley, Berkeley CA 94720 Department of Plant & Microbial Biology, University of California, Berkeley, Berkeley CA 94720; Department of 7 Chemical and Biomolecular Engineering and Department of Bioengineering, University of California, Berkeley, Berkeley CA 94720; Novo # Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kogle Alle´, DK2970-Hørsholm, Denmark Corresponding Author, [email protected]

Due to the rich history of Streptomyces as producers of important natural products, this genus of bacteria has recently garnered attention for its potential applications in the broader context of synthetic biology. However, the dearth of genetic tools available to control and monitor protein production precludes rapid and predictable metabolic engineering that is possible in hosts such as Escherichia coli or Saccharomyces cerevisiae. In an effort to improve genetic tools for Streptomyces venezuelae, we developed a suite of standardized, orthogonal integration vectors and an improved method to monitor protein production in this host. These tools were applied to characterize heterologous promoters and various attB chromosomal integration sites. A final study leveraged the characterized toolset to demonstrate its use in producing the biofuel precursor bisabolene using a chromosomally integrated expression system. These tools advance S. venezuelae to be a practical host for future metabolic engineering efforts. Keywords: Streptomyces, Bisabolene, Fluorescent Protein, Promoter The past two decades have witnessed a dramatic change in the scope and complexity of efforts within the space of synthetic biology. Accomplishments have progressed from the simple reconstitution of biosynthetic pathways in heterologous hosts to ambitious refactoring medicinally relevant compounds (e.g., artemisinin,

1-3

4-6

efforts that provided the ability to produce high titers of strictosidine,

replacements (e.g., bisabolene and fatty acid derivatives).

10-15

7

and taxadiene

8,

9

) or petrochemical

As synthetic biology applications have grown in

complexity, so too has the sophistication of available genetic and biochemical tools. Well characterized expression vector sets,

16

promoters for dynamic pathway control,

advanced methods for genome editing

22-25

17-19

tunable protein degradation

20, 21

and

represent a small fraction of the numerous characterized biological

parts and methodologies currently available in the synthetic biologist’s toolbox. Owing to their exemplary behavior, well mapped metabolic pathways, and the availability of many of the aforementioned genetic and biological components, synthetic biologists have often favored two organisms for the expression and optimization of heterologous biosynthetic pathways: Escherichia coli and Saccharomyces cerevisiae. Ideally, tools could be developed to allow the manipulation of any microorganism, but given the inability to culture a majority of single-celled organisms and the genetic intractability of many culturable bacteria, it seems that the development of methods to alter any given microbe is likely a distant reality. An alternate

1 ACS Paragon Plus Environment


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approach to expand the range of available hosts centers on the development of biological parts for model microorganisms, such as the cyanobacterium Synechococcous sp. strain PCC 7002, nidulans,

28

Geobacillus

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26, 27

the mold Aspergillus

or, ideally, a representative of the diverse actinobacterial phylum.

Due to its many favorable traits, such as the ability to perform consolidated bioprocessing, catabolize numerous hexose and pentose sugars, as well as a well documented existence of complex secondary metabolic pathways, recent, notable efforts have begun to focus on the development of Streptomyces (e.g., S. coelicolor, 31

2

12

30,

32

S. lividans, S. venezuelae, and S. albus ) as a chassis for routine synthetic biology applications. We have

specifically focused on S. venezuelae ATCC 10712 (hereafter referred to as simply S. venezuelae — when referring to the alternate S. venezuelae strain it will be identified with its unique ATCC 15439 number) as our host of choice due to its rapid doubling time (ca. 40 min), lack of mycelial clumping, sporulation in liquid media, and superior genetic tractability. In order to effectively bring the genetic toolbox for S. venezuelae and, in general, the broader class of actinobacteria to the level that current model microorganisms benefit from, well-characterized biological components and simple methods to assess and modulate protein production are required. Therefore, to increase the number of available biological tools and well-characterized genetic components for this host, we aimed to accomplish three goals in the current study: 1) identify a reporter that can be used in vivo to provide realtime assessment of factors that control protein production; 2) apply the optimized reporter system to characterize a set of orthogonal integration vectors that contain varied promoters and integration elements; and 3) demonstrate the use of these new biological tools to rapidly and markedly improve the production of bisabolene, a terpenoid natural product and biofuel precursor.

Results and Discussion Identification of an in vivo reporter for Streptomyces To develop next generation tools for S. venezuelae, it is critical that a reporter be available to allow readout of gene expression in a rapid and reliable manner with minimal disruption to the cell. One reporter system commonly employed in Streptomyces to assess factors that influence protein production (e.g., ribosome binding sites or promoters) relies on the enzymatic oxidation of catechol to 2-hydroxymuconic semialdehyde by the catechol dioxygenase XylE.

32, 33

This assay has numerous limitations, not the least of which are the lack of

real-time readout and the necessity to lyse cells prior to execution of the assay. These factors significantly lower


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the throughput and practicality of this method. Recent reports have described β-glucuronidase

34

and green

fluorescent protein (GFP) as an improved means to assess protein production in Streptomyces. Unfortunately, βglucuronidase based assays require substrate addition and its subsequent catalysis to initiate readout, a process that limits the real-time readout of activity, while GFP routinely exhibits a small dynamic range of fluorescence requires the formation of protoplasts (removal of the exterior cell wall) prior to analysis.

36

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or

To improve on these

assays we looked to further explore the use of fluorescent proteins (FPs) in intact cells as a means to assess the influence of promoter strength and ribosome binding site (RBS) sequences on gene expression. While FPs have been used to investigate spatiotemporal production of specific proteins in Streptomyces using microscopy,

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optimization of the fluorescence signal and data concerning the dynamic range of such an assay is lacking. Accordingly, a thorough screen of available FPs was executed to identify a candidate with desirable qualities to carry forward and characterize new genetic components for S. venezuelae. It is widely known that FPs possess dramatically differing intrinsic properties (i.e., excitation/emission wavelength, relative brightness, quantum yield and extinction coefficient) (Table S1), therefore screening a modest number of FPs was likely to yield a superior reporter. FPs were optimized for Streptomyces codon usage (Table S2) and initially inserted in a newly designed expression vector that integrates into the S. venezuelae chromosome at the bacteriophage VWB attB site and expresses the downstream gene under control of the well characterized ermE* promoter (PermE*). Cultures were grown in shake flasks containing breathable stoppers, as this was found to be important for chromaphore maturation (Figure S1), and measurements were taken every 24 h for three days. Unexpectedly, the majority of FPs did not show a significant increase in signal over the control strain, excluding mTFP and mCherry, which averaged an approximate 5-fold and 50-fold increase in fluorescence, respectively, relative to background (Figure S2). These results were at odds with Western blot analysis that revealed a majority of the expected fluorescent proteins were solubly expressed in S. venezuelae (Figure S3-6). In an effort to further examine what caused the relative absence of signal for the majority of the selected FPs, we explored the use of a stronger reporter to drive expression of these genes. Interestingly, replacement of PermE* with the robust glyceraldehyde-3-phosphate dehydrogenase promoter (Pgapdh(EL)) from Eggerthella lenta (EL) provided signals significantly over background for all queried FPs (Figure 1). Despite the significant increase in signal for all FPs using Pgapdh(EL), mCherry remained the superior reporter as was initially demonstrated with PermE*.



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One additional point of interest was the localization of the FP itself within the cell. While bulk fluorescence measurements made on the culture demonstrated reproducibility and low variance, we aimed to determine whether the signal was localized or present throughout the cell. Cells expressing mCherry under control of Pgapdh(EL) were examined under a high-power microscope (Figure 2); the fluorescence was observed to be distributed throughout the cell, which opens the door to specific biotechnology applications that use fluorescent proteins to gauge protein-protein interactions, protein localization or metabolite detection and readout in a high throughput manner.

Development of a set of orthogonal integration vectors for Streptomyces In an effort to enable rapid integration and predictable expression of heterologous biosynthetic pathways in S. venezuelae we aimed to leverage our recently established reporter system to characterize a number of bacteriophage integration sites that widely exist in Streptomyces spp. and describe the strength of expression at these sites using a suite of differing promoter/ribosome binding site (RBS) combinations. Bacteriophage-mediated integration of foreign DNA is known to occur through site-specific recombination at the attB locus located on the host chromosome and a corresponding attP site located on the integrating DNA, catalyzed by a specific, cognate integrase (Int). Pioneering work demonstrated the ability to target foreign DNA to the host chromosome (attB chromosomal site) by engineering conjugative plasmids to contain a suitable attP site and corresponding bacteriophage Int gene. sites, attB

VWB

, attB

ΦC31

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Given that many Streptomyces strains contain one, or more, of three common attB

and attB

ΦBT1 42-44

,

three vectors were designed to allow multiple, stable integrations in the

chromosome. To enable the integration of up to three heterologous DNA segments in a single host, a set of vectors were designed that could be conjugated from E. coli ET12567 into S. venezuelae, or any suitable host containing the cognate attB site. To facilitate conjugation and chromosomal insertion each plasmid was designed to contain the RP4 origin of transfer, one of the aforementioned attP sites, the corresponding Int gene, and a selectable antibiotic resistance marker. Further, each orthogonal vector set was designed to contain the base promoter PermE*, identical 5’- and 3’-untranslated regions (UTR) and a common segment containing multiple cloning sites to simplify the plasmid set and standardize expression. To enable rapid exchange of genetic elements though contemporary methods (e.g., Gibson assembly,

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CPEC

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or SLIC ), each plasmid was designed with the


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promoter and gene of interest flanked by unique blunt-cutting restriction sites (ScaI, FspI and EcoRV) (Figure 3). R

In total, three base vectors were designed, pAV (apramycin , attP R

R

(thiostrepton /beta lactam , attP

ΦBT1

VWB

R

), pSC (spectinomycin , attP

ΦC31

) and pTB

), to allow simultaneous integration at all three chromosomal attB sites. All

parent vectors were further diversified using fourteen additional promoters to substitute for PermE* that possess widely varied strengths (vide infra) to ultimately generate a suite of 45 vectors to be used in downstream engineering applications.

Analysis of natural and synthetic promoters at multiple attB loci We next desired to showcase our improved assay not only to characterize a suite of promoters and ribosome binding site (RBS) combinations, but also, for the first time, to describe expression at the three potential attB integration sites present in the S. venezuelae genome. The fifteen promoters mentioned above were assessed at the three attB sites, including 6 promoters identified from RNA Seq studies in S. albus (P13, P15, P1921,

and P27),

PSP05),

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4 from alternate actinobacteria (PermE*, Pgapdh(EL), PrpsL(RO), PkasO*) and 5 synthetic promoters (PSP01-

all previously demonstrated to cover a wide range of expression strengths in alternate Streptomyces

strains. All promoters were synthesized and inserted in our orthogonal vectors to drive expression of the mCherry gene. While hundreds to thousands of transconjugants per conjugation event were observed when the integrating vectors were directed to the VWB and ΦC31 attB sites, the ΦBT1 site proved to be relatively recalcitrant to integration. This result was not unsurprising as the initial report of the ΦBT1 site does not specify which S. venezuelae strain was used (ATCC 10712 vs. 15439)

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and, unlike the ATCC 15439 strain, the ATCC

10712 strain lacks the strictly conserved ΦBT1 site (potential pseudo-sites are present).

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Moreover, not only

were transconjugants using the ΦBT1 integration site rare, likely resulting from integration at a pseudo-attB site, but also the few clones that were obtained exhibited impaired growth characteristics and attenuated fluorescence (approximate 90% decrease in fluorescence for resultant strains compared to VWB). Given these results, no further work with ΦBT1 integrating vectors was conducted. Initial inspection of the fluorescence data revealed two major trends with regard to relative fluorescence levels between the two viable sites (i.e., attB

VWB

and attB

ΦC31

). The first group of promoters (Figure 4A) appeared

to be slightly stronger at the ΦC31 attB site than at VWB attB — routinely exhibiting a 10-20% increase in



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fluorescence. These promoters include PermE*, Pgapdhp(EL), PrpsLp(RO) and native promoters recently identified from S. albus.

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Interestingly, there appears to be a second cluster of promoters to emerge from the data that showed an

inverse trend compared to the previous data set. This group, which largely consists of the synthetic promoters (i.e., PSP01-PSP05), appears to have significantly attenuated activity at the ΦC31 site compared to VWB. The result is an average reduction in fluorescence at ΦC31 vs. VWB on the order of 35-45% (Figure 4B). Synthetic promoters PSP01-PSP05 have been modularly assembled through the combination of a synthetic promoter, ribosome binding site (RBS) and the riboJ insulator as reported by Bai et al. the 5’ untransulated region of mRNA prior to translation

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The riboJ insulator is widely known to cleave

and, therefore, the reduction in signal at the ΦC31 site

may originate from the cleavage of a promoter and/or RBS upstream of the engineered RBS for mCherry and result in diminished protein production. Curiously, PkasO*, a well-known strong promoter, also shows similar features to the synthetic promoters, despite not having a known 5’ mRNA cleaving ribozyme encoded in the sequence. Ultimately, these empirical data suggest that at least two groupings of promoters exist and inspection of new promoter/RBS combinations is suggested for metabolic engineering efforts if precise control of protein levels or metabolic flux is necessary. Combinatorial Improvement of Bisabolene Production Using Chromosomally Integrated Genes While previous aims in this study were centered on the design and assessment of tools for biosynthesis, one of our ultimate goals was to employ these tools to rationally and markedly improve the production of specialized metabolites. Previous efforts have demonstrated the heterologous production of bisabolene in S. venezuelae,

12

but due to the lack of well-characterized biological tools our initial system relied on a production

scheme that was sub-optimal for industrial applications. The first shortcoming centered on the fact that the system employed a replicating plasmid to express the bisabolene synthase gene coAgBis. Replicating plasmid vectors have many drawbacks such as instability, variances in intracellular copy number, and the requirement for antibiotic to maintain selective pressure on plasmid retention – an undesired addition to operating expenses when attempting to generate low-cost bioproducts. In addition, although the second gene required to increase bisabolene production, farnesyl pyrophosphate synthase (FPPS), was integrated at the VWB site, only the PermE* was tested to drive its expression. The ability to use stronger promoters to drive FPPS expression provided a significant opportunity to increase flux through this pathway. Hence, while the original system was effective in demonstrating the first heterologous production of this biofuel precursor in Streptomyces, there existed


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opportunities to improve parameters that could both lower production costs and make this system more amenable to industrial application. Our attempt to optimize bisabolene production using a chromosomally integrated system focused on the two genes explored in the aforementioned study: FPPS and coAgBis, the bisabolene synthase gene from Abies grandis codon optimized for S. venezuelae (Figure 5).

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Rather than simply use the strongest promoters to drive

expression of each gene, we aimed to build a small combinatorial library using promoters of varied strengths to 1) determine whether production titer scales with increase of promoter strength and 2) investigate if bisabolene production could reach production levels that are toxic to the cells. Therefore, use of promoters that possess a wide range of strengths could potentially provide an improved strain compared to one designed by simply using the strongest promoters. Both coAgBis and FPPS were inserted into the integrating vectors pAV and pSC, respectively, with promoters that possessed activities ranging from moderate to very strong (i.e., PermE*, P20, Pgapdh(EL), PkasO*, PSP01). Given that FPPS was shown to be critical in increasing bisabolene titers, we opted to limit promoters driving expression to the four strongest listed, thus eliminating PermE*. The vectors were integrated in S. venezuelae to generate a library of 20 bisabolene producing variants. The resultant strains were screened in 5 mL culture tubes and, while this culture condition is sub-optimal for bisabolene production, the results revealed a strong correlation between the promoter strength and titer achieved. Inspection of the data clearly revealed that strains expressing coAgBis and FPPS under control of PkasO* and/or PSP01 generated the highest titers, with titers ranging from 40 µg/L to 1.77 mg/L (Table 1). Moving forward we tested the four best variants against our previously constructed bisabolene production strains, S. venezuelae SZ01 and SZ04.

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The first generation bisabolene producing strain, S. venezuelae SZ01,

employed PermE* to drive expression of coAgBis from a high copy replicating vector (a pUWL-oriT derivative containing the pIJ101 origin of replication).

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Improvement on this system resulted in S. venezuelae SZ04,

which uses the same expression system for coAgBis, but also expresses a second copy of FPPS1 using an integrated plasmid, similar to that of pAV-1, at the VWB site. The best four strains constructed using only chromosomal-based expression of both genes from Table I, along with S. venezuelae SZ01 and SZ04, were grown under shake flask conditions in PM-4 medium,

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and assessed for bisabolene production. Titers ranged

from 0.71 to 5.38 mg/L (Table 2), with S. venezuelae RP01 (PkasO*p-coAgBis::VWB; PkasO*p-FPPS1::φC31) showing the greatest production. The strains using fully integrated genes compared well to both S. venezuelae



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SZ01 and SZ04, which produced 2.94 and 9.41 mg/L, respectively. Given that S. venezuelae SZ01 benefits from the use of a replicating vector (pIJ101 origin of replication), which is estimated to be on the order of 40 – 300 copies per cell,

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it is not surprising that we observed a modest reduction (ca. 40%) in titer when comparing the

best production strain SZ04 to RP01 that used single copies of the two integrated genes. Taken together, our results clearly demonstrate the ability to perform combinatorial integration at two attB sites and generate significant product titers, despite relying on single copies of a given gene. These data not only set the stage for future combinatorial engineering efforts, but also clearly positions this host for specialty metabolite production under conditions that better resemble those used in industry.

Conclusion In an effort to improve the biological tools available for the model Streptomycete S. venezuelae, we screened and identified an optimal FP to provide real-time readout of protein production in intact cells. This improved assay was not only used to determine protein production resulting from heterologous promoter and RBS combinations, but also to investigate expression at different attB sites in the same host chromosome. We generated 45 expression vectors that can simultaneously integrate at three different attB sites and characterized 15 promoters that possess relative strengths that vary over three orders of magnitude. Results illuminated key differences at the φC31 and VWB attB sites, an important factor when attempting to properly balance heterologous metabolic pathways. Ultimately, the tools and information generated were used to develop a chromosomally integrated bisabolene production pathway that produced 5.4 mg/L. This value, while being generated from a chromosomally integrated expression system, compares well to previous S. venezuelae bisabolene production strains that rely on autonomously replicating vectors. When applied to biosynthetic pathways our results clearly demonstrated that the newly validated mCherry assay translates well to alternate systems (Table I) where secondary metabolism (i.e., bisabolene biosynthesis) could be incrementally and reliably improved based on the combinatorial use of promoters characterized in this study. In summary, the biological tools developed here serve to streamline the introduction or modification of biosynthetic pathways in S. venezuelae and, in general, the broader genera of Streptomyces by allowing more predictable protein production and simplifying the introduction of multiple genes or operons. Accordingly, these efforts set the foundation for the


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production of numerous natural and rationally altered secondary metabolites, many of which have important uses in modern medicine or industry.

Materials and Methods Measurement of S. venezuelae fluorescence Fresh S. venezuelae spores were used to inoculate 10 mL of tryptic soy broth, which contained both the appropriate antibiotic to select for the integrated vector and naladixic acid (30 µg/mL). Cultures were grown to saturation overnight at 30 °C and used to inoculate (3% vol/vol) 30 mL of MYM broth contained in 250 mL Erlenmeyer flasks fitted with breathable, foam stoppers. All variants were grown in triplicate culture at 30 °C for 72 h and measured every 24 h. For fluorescence measurements, each sample was collected (500 µL), pelleted and washed with sterile ddH2O (2 x 1 mL). The final pellets were resuspended in sterile ddH2O (500 µL) and used immediately for measurement. Fluorescence was read at the appropriate λex/em as indicated in Table S2 using a Biotek Synergy 2 plate reader (BioTEk, Winooski, VT). Fluorescence was normalized to cell density as determined by the OD600 reading of cells prepared at a 1:20 dilution. Increase in fluorescence was determined by the increase of a given strain over a control strain containing an integrated vector lacking the desired fluorescence gene.

Generation of vectors expressing fluorescent proteins Gene blocks were designed for each fluorescent protein using the IDT (Integrated DNA Technologies (IDT), Coralville, IA) codon optimization tool to recode each fluorescent protein using S. coelicolor A3(2) codon usage (Table S2). To allow for Gibson assembly, each gene was designed with a 30 base pair region of homology to pAV-Pgapdh(EL) at the 5’- and 3’ ends. The recipient plasmid (pAV-Pgapdh(EL)) was digested with FspI and EcoRV to provide a linearized vector backbone, which was purified using gel electrophoresis. The DNA segment containing the fluorescent protein of interest and FspI/EcoRV digested pAV-Pgapdh(EL) were mixed in a 1:1 ratio and subjected to Gibson assembly

45

under standard conditions. DNA was introduced into E. coli DH10B by transformation.

Colonies were selected and plasmids were sequenced to verify the correct plasmid construction.



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Exchange of promoters in the integrating vectors Gene blocks were synthesized according to the promoter/ribosome binding site (RBS) to be used (Table S4). Any DNA segments lacking homology to the destination vector (see Tables S3 and S4) were amplified with PCR oligomers that introduced 30 base pair regions of homology at the 5’ and 3’-ends to enable Gibson assembly between the promoter/RBS DNA and the target vector (pAV, pSC or pTB). The promoter/RBS DNA was mixed in 45

a 1:1 ratio with the linearized vector (cut with FspI and ScaI) under standard Gibson assembly conditions.

DNA

was introduced into E. coli DH10B by transformation. Colonies were selected and plasmids were sequenced to verify the sequence was that of the plasmid of interest.

Conjugal transfer of vectors to S. venezuelae E. coli ET12567 was transformed with the appropriate vector (derivatives of pAV, pSC, or pTB) and selected for on LB agar containing chloramphenicol (15 µg/mL), kanamycin (25 µg/mL) and either apramycin (25) µg/mL, spectinomycin (50 µg/mL) or carbinecillin (100 µg/mL), depending on the vector employed. A single colony was used to inoculate a 5 mL overnight culture containing antibiotics as outlined above. The overnight culture was used to seed 50 mL of LB, again charged with the appropriate antibiotics which was grown to an OD600 of 0.4-0.6. The culture was pelleted and washed with LB twice and finally resuspended in 5 mL LB. Fresh S. venezuelae spores were collected from a plate with 5 mL 2xYT and incubated at 50 ºC for 10 minutes. The spores (0.5 mL) and E. coli cells (0.5 mL) were mixed, spread on mannitol soy agar (MS) and allowed to incubate at 20 ºC overnight (approximately 16 h). Following incubation, naladixic acid (30 µg/mL), and the selective antibiotic, apramycin (25) µg/mL, spectinomycin (50 µg/mL) or thiostrepton (30 µg/mL), were applied. Plates were incubated for three days, at which point colonies were transferred to fresh, selective MS agar plates. PCR was used to validate the resultant colonies for integration of the vector at the appropriate attB site and in each case it was confirmed that the pAV and pSC vectors do selectively integrate at the appropriate attB site (Figure S7-8).

Small Scale Culture and Analysis of Bisabolene Production Twenty strains possessing coAgBis integrated and FPPS1 integrated at the VWB and ΦC31 sites, respectively, were streaked out on glycerol-arginine agar and let sporulate. Throughout this experiment the appropriate antibiotics were used when required. Fresh spores were used to inoculate 5 mL of tryptic soy broth and grow at


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30 ºC shaking at 200 RPM overnight. The cultures were then used to seed production cultures (3% vol/vol inoculation, done in biological triplicate), which consisted of 5 mL of MYM media (see Supplementary Information) in a 30 mL culture tube. Cultures proceeded to grow for 24 h at 30 ºC with shaking at 200 RPM before a 10% overlay of decane was added. The cultures were let proceed at the aforementioned temperature for an additional 48 h. The decane overlay was isolated and analyzed by GC-MS using a method previously described.

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Bisabolene titers were compared to a standard curve of authentic bisabolene.

Increased Scale Culture and Analysis of Bisabolene Production Improved production of bisabolene was performed exactly as started for the small-scale production with two notable exceptions. First, production took place on the 50 mL scale in 250 mL Erlenmeyer flasks. Secondly, a 20% decane overlay was used as opposed to the previously stated 10%.

S. venezuelae microscopy All strains were prepared for microscopy as stated above for fluorescence measurements and prepared as a wet mount. Each strain was imaged on a Leica DMC 4000 microscope using a Hamamatsu ORCA flash-4.0LT camera using a 63x objective. Images taken using visible light were done using differential interference contrast (DIC). Fluorescence images were obtained using the Leica TX2 filter cube.

Supporting Information. Figures S1-S9 (supporting main text), Tables S1-S4 (supporting main text), additional experimental protocols and media compositions are described in the Supplementary Information.

Acknowledgements We would like to acknowledge W. Moore for assistance in microscopy studies. This work was funded by the National Science Foundation Catalysis and Biocatalysis Program (CBET-1437775), the Department of Energy EERE Annual Operating Plan (BM0101020-05450-1004171/Agreement 28712/DE-AC02-05C11231), and by the DOE Joint BioEnergy Institute (http://www.jbei.org), which is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between



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Figure 1. Screen of seven differing fluorescent proteins integrated at the VWB attB site under the control of strong, constitutive glyceraldehyde-3-phosphate dehydrogenase promoter (Pgapdhp(EL)) from Eggerthella lenta (EL). Samples were taken at 24, 48, and 72 hours and normalized to optical density.



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Figure 2. Microscope images of S. venezuelae containing mCherry under control of the PSP01 promoter at the VWB attB site (top, A and B) compared to the wild-type strain (bottom, C and D). Left) Comparison of the mCherry-containing and wild-type strains under visible light using differential interference contrast (DIC); Right) Comparison of fluorescence using the TX 2 filter for mCherry visualization.


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Figure 3. Generic map of the base vectors generated for simultaneous integration with key features noted. Each vector contains identical 5’and 3’-untranslated regions flanking the promoter and gene as well as unique restriction sites to introduce new genetic elements. Resistance R R markers, integrase genes and the corresponding attP sites all differ on pAV (apramycin , VWB), pSC (spectinomycin , ΦC31) and pTB R R (thiostrepton /beta lactam, ΦBT1) to enable concurrent integration. Available promoters with differing expression strengths are listed in Figure 4A and 4B.



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Figure 4. Fluorescence data from the analysis of fifteen differing promoters at two integration sites. A) Promoters that exhibit moderately stronger expression at the ΦC31 site compared to VWB and B) Promoters that cause a significant decrease in expression at the ΦC31 site compared to VWB. Samples were taken at 24, 48, and 72 hours and normalized to optical density.


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Figure 5. Scheme for bisabolene production in S. venezuelae. Both FPPS and coAgBis are inserted at the ΦC31 and VWB sites, respectively, and expressed by promoters of varying strength.



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Table 1. Small scale screening of bisabolene producing strains. Bisabolene production ranged from not detected (n.d.) (red) to approximately 2.0 mg/L (green).


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Table 2. Shake flask production of bisabolene with selected high-titers strains as compared to previous generation strains (SZ01 and SZ04), which relied on the coAgBis gene being expressed from a replicating vector (pIJ101 derivative) and not integrated in the chromosome as with RP01-04.



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Development of Next Generation Synthetic Biology Tools for Use in

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