Engineering Streptomyces coelicolor for production of monomethyl branched chain fatty acids

Journal Pre-proof Engineering Streptomyces coelicolor for Production of Monomethyl Branched Chain Fatty Acids Jeong Sang Yi, Hee-Wang Yoo, Eun-Jung Kim, Yung-Hun Yang, Byung-Gee Kim

PII:

S0168-1656(19)30908-3

DOI:

https://doi.org/10.1016/j.jbiotec.2019.10.020

Reference:

BIOTEC 8537

To appear in:

Journal of Biotechnology

Received Date:

24 July 2019

Revised Date:

24 October 2019

Accepted Date:

29 October 2019

Please cite this article as: Yi JS, Yoo H-Wang, Kim E-Jung, Yang Y-Hun, Kim B-Gee, Engineering Streptomyces coelicolor for Production of Monomethyl Branched Chain Fatty Acids, Journal of Biotechnology (2019), doi: https://doi.org/10.1016/j.jbiotec.2019.10.020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Engineering Streptomyces coelicolor for Production of Monomethyl Branched Chain Fatty Acids

Jeong Sang Yia,1, Hee-Wang Yooa,b, Eun-Jung Kima,c, Yung-Hun Yangd,e, and Byung-Gee Kima,f*

bInterdisciplinary

of

of Molecular Biology and Genetics, Seoul National University, Seoul, South Korea Program for Biochemical Engineering and Biotechnology, Seoul National

ro

aInstitute

University, Seoul, Republic of Korea Institute, Seoul National University

dDepartment

of Biological Engineering, College of Engineering, Konkuk University, 1

re

Hwayang-dong, Gwangjin-gu, Seoul, 143-701, Korea

for Ubiquitous Information Technology and Applications (CBRU), Konkuk University,

Seoul 143-701, South Korea

of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea

1Present

ur na

fSchool

lP

eInstitute

-p

cBio-MAX

Address: Center for Marine Biotechnology and Biomedicine, Scripps Institution of

Oceanography, University of California San Diego, 9500 Gilman Drive 0204, La Jolla,

Jo

California, USA, 92093-0204

*Correspondence: Byung-Gee Kim, School of Chemical and Biological Engineering, Seoul National University, 1, Gwanak-ro, Gwanak-gu, Seoul 08826, Republic of Korea; E-mail: [email protected], Phone: +82-2-880-6774, Fax: +82-2-876-8945

Highlights    

Leucine is the most effective nutrient for Branched Chain Fatty Acid production Removal of polyketide antibiotics production enhanced fatty acid productions bkdABC overexpression is the most effective in BCFA production 354.1 mg/L of BCFA, 90.3 % of total FA, were produced from Streptomyces coelicolor

of

Abstract Branched chain fatty acids (BCFA) are an appealing biorefinery-driven target of fatty acid (FA)

ro

production. BCFAs typically have lower melting points compared to straight chain FAs, making them useful in lubricants and biofuels. Actinobacteria, especially Streptomyces species, have

-p

unique secondary metabolism that are capable of producing not only antibiotics, but also high

re

percentage of BCFAs in their membrane lipids. Since biosynthesis of polyketide (PK) and FA partially share common pathways to generate acyl-CoA precursors, in theory, Streptomyces sp.

lP

with high levels of PK antibiotics production can be easily manipulated into strains producing high levels of BCFAs. To increase the percentage of the BCFA moieties in lipids, we redirected

ur na

acyl-CoA precursor fluxes from PK into BCFAs using S. coelicolor M1146 (M1146) as a host strain. In addition, 3-ketoacyl acyl carrier protein synthase III and branched chain α-keto acid dehydrogenase were overexpressed to push fluxes of branched chain acyl-CoA precursors towards FA synthesis. The maximum titer of 354.1 mg/L BCFAs, 90.3 % of the total FA moieties,

Jo

was achieved using M1146dD-B, fadD deletion and bkdABC overexpression mutant of M1146 strain. Cell specific yield of 64.4 mg/L/gcell was also achieved. The production titer and specific yield are the highest ever reported in bacterial cells, which provides useful insights to develop an efficient host strain for BCFAs.

Keywords: Branched Chain Fatty Acids, Streptomyces coelicolor

1. Introduction As populations continue to grow, renewable fuels will be in greater demand to offset

of

anthropogenic demands that are straining natural carbon resources (Xu et al., 2017). Biofuels

ro

have been appealing targets of renewable and sustainable energy sources. Metabolic engineering of microbial production pathways of such biofuels is a very trendy topic of research in the oil

-p

industry (Koppolu and Vasigala, 2016). Biodiesels comprising branched chain fatty acid (BCFA) esters attracted great attention, owing to high performing chemical properties over that

re

comprising only straight chain fatty acid (SCFA) esters. BCFA esters are preferred biofuels for

lP

uses in cold environments because they have lower melting points (MP) than that of SCFA esters (Yao and Hammond, 2006). Palmitate methyl ester has a MP of 30.7 ℃, while isopalmitate

ur na

methyl ester has a MP of 18.2 ℃. BCFA esters also serve as better lubricants because viscosities of BCFA esters are also lower than those of SCFA (Bart et al., 2012; Zhang et al., 2004). Due to such advantages, recent studies began to focus on engineering microbes to produce BCFAs. Above all, branched chain amino acid (BCAA) catabolism was the major target of metabolic

Jo

engineering, because their degradation pathways supply both straight chain and branched chain acyl precursors for SCFA and BCFA biosynthesis (Kim et al., 2014). The pathways also provide fatty acid (FA) chain elongation units at the same time. Isobutyryl-CoA, isovaleryl-CoA, and 2-methylbutyryl-CoA are generated as degradation products of valine, leucine, and isoleucine, respectively. These three precursors become starter units for BCFA synthesis (Kaneda, 1991). Each precursors leads to a different iso-form of

BCFAs: isobutyryl-CoA becomes iso-form even-chain BCFAs, isovaleryl-CoA becomes iso-form odd-chain BCFAs, and 2-methylbutyryl-CoA becomes anteiso-form odd-chain BCFAs (Body, 1984). These branched chain acyl-CoAs bind to 3-ketoacyl acyl carrier protein synthase III (fabH) of a fatty acid synthase (FAS), which then undergo repetitive chain elongation by incorporation of malonyl-CoA (Revill et al., 1996) (Fig. 1). SCFAs are mainly synthesized from an acetyl-CoA as a starter unit, which only requires central carbon metabolism. As a result,

of

manipulation of a BCAA degradation pathway would be a key to increase the production of

ro

BCFAs.

E. coli is a widely used host for FA production due to its plentiful, easy to use genetic

-p

tool boxes for gene manipulations. However, E. coli only produces SCFA because E. coli fabH

re

only accepts acetyl-CoA and malonyl-CoA as substrates (Choi et al., 2000), which only requires central carbon metabolism. Therefore, fabH and branched chain α-keto acid dehydrogenase

lP

(bkdABC) from other microorganisms were heterologously expressed in E. coli to harness the catabolic pathways of BCAAs and produce BCFAs (Bentley et al., 2016; Cropp et al., 2000;

ur na

Haushalter et al., 2014). In search of a host strain to minimize such pathway reconstructions, Streptomyces species attracted our attention since the strains already produce and consume BCFAs.

Up until now, Streptomyces species were known to be excellent hosts for secondary

Jo

metabolite production, some of which are polyketide (PK) antibiotics and anticancer drugs whose backbone sometimes comprise BCFA moieties (McKenzie et al., 2010). Streptomyces are an ideal candidate as host strains for BCFA production because BCAA degradation pathways have been well characterized (Stirrett et al., 2009). Moreover, about 70 ~ 75 % of entire fatty acid pools in Streptomyces coelicolor are composed of branched chain fatty acids (Li et al., 2005).

The most abundant fatty acid among them is isopalmitate (iC16), accounting for about 20% of total FAs. As a result, there are no need for extra pathway reconstructions or heterologous expressions of foreign genes. Enhancing the production of FA requires increasing supplementations of both starter and extender acyl-CoA units. Our previous studies to increase production of PK antibiotics showed that Streptomyces strains have a great potential for BCFA production. Overexpressing bkdABC in

of

S. coelicolor increased the production of PK antibiotics actinorhodin (ACT) by 52 folds of the

ro

wild type (WT) (Kim et al. 2014). As ACT is composed of 2 acetyl-CoAs and 14 malonyl-CoAs, bkdABC overexpression was shown effective for supplementations of both starter and extender

-p

units.

re

Since PK antibiotics and FAs share common acyl-CoA precursor pools (Brakhage, 2004; Tanaka et al., 2017; Yi et al., 2018), applying the same gene engineering strategies from

lP

PK overproduction should also increase FA production in Streptomyces sp.. In this study, we developed S. coelicolor as a host strain for BCFA production by pushing, pulling, and redirecting

ur na

acyl-CoA fluxes. bkdABC was overexpressed to accelerate BCAA catabolism, and fabH overexpressed to initiate FA synthesis (Fig. 1). Acyl-CoA fluxes from PK synthesis redirected toward FA synthesis by using S. coelicolor M1146 strain, which had deletions of four major secondary metabolite synthesis gene clusters. Here we converted PK antibiotics producing S.

Jo

coelicolor into a biofuel production host for the first time.

2. Materials and Methods 2.1 Bacterial strains, plasmids, and culture conditions Plasmids and cosmids used throughout the study are described in Table 1, and bacterial strains

are listed in Table 2. For cultures of E. coli strains, Luria-Bertani (LB) Broth (Becton Dickinson, USA) was used along with appropriate antibiotics when necessary. R5- complex medium made by standard protocols (Kieser et al., 2000) were used for cultures of S. coelicolor M1146 and its mutants. Difco Nutrient Agar (DNA) medium (Becton Dickinson, USA) and Mannitol Soya flour (MS) agar media, containing 20 g/L mannitol, 20 g/L soya flour, 20 g/L agar in 1 L of distilled water, were used for conjugations of cosmids into S. coelicolor from E. coli ET12567.

of

Appropriate antibiotics were also used for selections of mutants. Antibiotics were used in

ro

concentrations of 0.1 mg/mL ampicillin, 8 μg/mL thiostrepton, and 50 μg/mL apramycin. For removals of E. coli from conjugated S. coelicolor, 25 μg/mL nalidixic acid was used for cultures

-p

on agar plates.

re

E. coli and S. coelicolor strains were cultured on Lab Companion SK-71 Benchtop shaker (Jeio Tech, South Korea), at 37 ºC and 30 ºC with 200 rpm, respectively. For S. coelicolor

lP

strains, 200 mg of wet weighted cells from overnight 50 mL R5- seed cultures in 250 mL flasks were inoculated to fresh 50 mL R5- media for production of both ACT and BCFAs. Final 2 mM

ur na

of valine, leucine, and isoleucine were added at this point when needed. 5 mL of cell sample cultures were harvested and dried in a 60 ºC oven for measurement of dry cell weights (DCW).

2.2 Strain constructions

Jo

Sequence information of all the primers used in this study is described in Table S1. Deletion cosmid for actII-orf4, SCO5085, was constructed using pSupercos1 vector. Primers 1 and 2 were used to PCR amplify upstream region of actII-orf4. Primers 3 and 4 were used to clone LoxP and apramycin resistance gene using pIJ773 as a template. Primers 5 and 6 were used for downstream region of actII-orf4.

fabH, SCO2388, was PCR amplified with primers 7 and 8, and was inserted into PstI and XbaI sites of pIBR25, resulting pIBR25::fabH. bkdABC, SCO3815 ~ 3817, was amplified with primers 9 and 10, and was inserted into XbaI and EcoRI sites of pIBR25 and pIBR25::fabH, resulting pIBR25::bkdABC and pIBR25::fabH::bkdABC, respectively. fadD, SCO6196, deletion cosmid was constructed using St2G5.17 cosmid. Deletion cassettes for fadD was constructed using primers 11 and 12, and pIJ773 as a template for

of

apramycin resistance marker. Deletion of actII-orf4 and fadD was performed according to a

ro

standard protocol (Kieser et al., 2000). Apramycin resistance marker was removed from cre recombinase gene, using pUWLCRE plasmid.

-p

ilvBC, SCO5512 ~ SCO5514, was PCR amplified using primers 13 and 14, and ilvD,

re

SCO3345, was amplified using primers 15 and 16. ilvBC was inserted into XbaI and EcoRI sites, and ilvD into EcoRI and HindIII sites of pIBR25, creating pIBR25::ilvBCD. poxB, SCO6155,

lP

was amplified using primers 17 and 18, and was inserted into BamHI and pstI sites of pIBR25, resulting pIBR25::poxB. Protoplast transformation was performed according to the standard

ur na

protocol (Kieser et al., 2000).

2.3 Fatty acids extraction and quantification From 50 mL of shake flask cultures, 5 mL samplings were made on day 6.200 μL acetic acid was

Jo

added to each sample, and 10 mL of 1:1 chloroform to methanol solution was subsequently added for extraction of FAs and lipids from S. coelicolor. Aqueous layer was removed from the samples, and the left over organic layer FAs was completely dried. The FA extracts from the organic layer were then reconstituted with 1mL of 5% H2SO4 in methanol for methyl esterification. 6.75 μL of 10 mM C17 FA were added to the samples at this point as an internal

standard. Replicate experiments were always performed using isogenic strains. Methyl esterification was performed by heating up the samples for 2 hours at 90 ºC in sealed vials. Fatty acid methyl esters (FAME) were then extracted with 200 μL hexane after 1 mL of 0.9% NaCl in water was added to the methyl esterified samples. All the authentic fatty acids for drawing out quantification curves were purchased from Sigma-Aldrich, USA. FAMEs were analyzed and quantified by GC-MS, a Trace GC Ultra Gas Chromatograph (Thermo

of

scientific, USA) connected to an ion trap mass detector, ITQ1100. TR-5MS column with 30 m X

ro

0.25 mm ID X 0.25 μm film (Thermo scientific, USA) was used for separation of FAMEs. GC oven started its temperature at 50 ℃, held for 1 min, and applied an increasing ramping rate of

-p

20 ℃/min up to 250 ℃, then held at 250 ℃ for 10 min before a new cycle began. It successfully

lP

peak identification and quantification.

re

separated FAMEs (Fig. S1). Fatty acid profiles were compared to that of authentic samples for

ur na

3. Results and discussion

3.1 Production of BCFA from ACT repressed mutant and BCAA supplementation Supplying methyl-branched acyl-CoAs from degradation of BCAAs is one of the most important factors in BCFA production. Since S. coelicolor is already known to produce about 50% of acyl-

Jo

CoA precursors from BCAA catabolism (Stirrett et al., 2009), we chose S.coelicolor M1146 to take advantages of its well-developed BCAA metabolism. Since we hypothesized that increasing acyl-CoA fluxes into FAS while reducing the fluxes towards PKS may result in overproduction of desired BCFAs, simple “push” and “pull” methodologies (Fig. 1) were used as previously described (Kim et al., 2014; Yi et al., 2018). First, we examined how deletion of a positive regulator affected FA biosynthesis and

BCFA content in PK production. ACT is the most highly produced PK antibiotics in S. coelicolor (Kim et al., 2014), and its production is regulated by several factors such as Sadenosylmethionine and bld sigma factor (Okamoto et al., 2003). Among them, deletion of actIIorf4, ACT pathway specific positive transcriptional regulator (Arias et al., 1999), was previously reported to reduce ACT production without affecting any cell morphology (Zhang et al., 2009). Indeed, ACT production from actII-orf4 deletion mutant (SCOdA) decreased down to 1.7 folds of

of

the WT (Fig. S2). We supplemented final 2mM concentrations of leucine, valine, and isoleucine

ro

to cultures of SCOdA to examine their effects on BCFA production (Fig. 2A). Starting units are directly produced from the three BCAAs that we wanted to examine their effects on BCFA

-p

production. One notable result was that leucine feeding reduced a lag phase of the cell growth,

re

resulting initial high cell OD (Fig. 2B). Biosynthesis of iC16 and C16 were increased in SCOdA compared to that of the WT, resulting in increased total FA production from 53.1 mg/L to 72.0

lP

mg/L. However, there were no big differences in BCFA distributions (Fig. 2C). The most FA was produced when leucine was supplemented, 198.5 mg/L with 93 % of BCFA contents, while that

ur na

of SCOdA was 72.0 mg/L with 60 % of BCFA contents. In terms of BCFA distributions, 51 %, 21 % and 14 % were iC16, aiC15, and iC15, respectively (Fig. 2D). Isoleucine and valine supplementation resulted total FA production of 56.7 mg/L with 84.8 % BCFA content, and 96.7 mg/L with 91.9 % BCFA content respectively. Such differences in BCFA production may be

Jo

resulted from enhanced growth in leucine supplemented cultures, but it requires more detailed studies to explain. BCAA metabolism is regulated by codY and bkdR, and it is very complex. The two regulators are present, but not well characterized in S. coelicolor. They are reported to control the BCAA metabolism in B. subtilis (Bergara et al., 2003; Brinsmade et al., 2010). Both codY and bkdR are regulated by, and respond differently towards each BCAAs, making a

complex regulatory network of BCAA metabolism. In our tested conditions, leucine was the most promising additives to cultures for BCFA production.

3.2 BCFA production from S. coelicolor M1146 Deletion of actII-orf4 improved the total FA production attributed by increases in iC16 and C16 biosynthesis in S. coelicolor A3 (2) M145 strain. We selected S. coelicolor M1146 (M1146) to

of

further increase BCFA production. M1146 has deletions of four different secondary metabolite

ro

gene clusters (Coze et al., 2013), i.e. ACT, undecylprodigiosin (RED), calcium-dependent antibiotic (CDA), and a cryptic type I polyketide synthase (CPK). M1146 was originally

-p

constructed as a host for heterologous gene expressions of other secondary metabolites (Gomez-

re

Escribano and Bibb, 2010). However, M1146 was also the best suitable strain for BCFA production, since most of acyl-CoA competing pathways were deleted.

lP

We carried out the experiments in both SCOdA and M1146 strains to select more suitable production host. First, we compared FA production of the WT, SCOdA, and M1146 strains. Cell

ur na

growth of M1146 somewhat decreased, but final cell mass of M1146 at the time of FA extraction was not very different from that of the WT and SCOdA (Fig. 3A). On the other hand, production of FAs in M1146 increased dramatically. 184.5 mg/L of FA with 90.2 % of BCFA contents were produced in M1146 (Fig. 3B). It was about 2.9 folds increase compared to that of the WT. Two

Jo

major FAs in M1146 were iC16, 45%, and aiC15, 29 % (Fig. 3C). Further experiments were carried out in M1146, since it was confirmed to produce more BCFAs than the other two could.

3.3 Utilizing BCAA catabolism for BCFA production Major differences between BCFA nonproducing bacteria and S. coelicolor come from fabH and

bkdABC, the first enzyme in fatty acids biosynthesis pathway, and the most important genes in BCAA catabolism, respectively. For example, BCFA nonproducing fabH only accepts acetylCoA, malonyl-CoA, and propionyl-CoA (Choi et al., 2000). As a result, alterations in fatty acid profiles from SCFA to BCFA or vice versa required cloning of B. subtilis fabH into E. coli (Jiang et al., 2015), or E. coli fabH to S. coelicolor (Li et al., 2005). Since Streptomyces sp. already have abilities to produce BCFAs, there were no needs for heterologous expressions of fabH and

of

bkdABC genes or reconstruction of BCAA metabolism. Having such advantages, we simply

ro

overexpressed these genes in S. coelicolor mutant strains, and their fatty acid production were compared to each other.

-p

All three M1146 mutants carrying pIBR25::fabH (M1146-F), pIBR25::bkdABC

re

(M1146-B), or pIBR25::fabH::bkdABC (M1146-FB) showed decreases in cell growth (Fig. 4A). Only original M1146 showed similar cell growth with that of the WT. The most significant

lP

decrease in DCW was observed in M1146-B, i.e. 6.2 g/L, which was only a half of the WT DCW. The maximum production of FAs was achieved with M1146-FB, i.e. 233 mg/L FAs and 86.9 %

ur na

of BCFAs (Fig. 4B). Production of FAs in M1146-F and M1146-B were almost identical to that of M1146, 170.5 mg/L with 89.9 % BCFA content, and 154.0 mg/L with 87.1 % BCFA content respectively. But cell specific yield was the highest in M1146-B, which was about 4.0 fold increases compared to that of the WT (Fig. 4C).

Jo

iC16 was the most pronounced FAs among the others, accounting about 50 % of the total

FAs in S. coelicolor. It is due to substrate specificities of S. coelicolor fabH, since fabH showed the highest isobutyryl-CoA substrate specificity among the three BCFA -ketoacyl-CoA precursors. (Smirnova and Reynolds, 2001). While iC16 and C16 were the two most abundant FAs in S. coelicolor A3 (2) M145 (Li et al., 2005), substantial amounts of aiC15, and decreases

in C16 concentrations were observed in all our tested M1146 strains. It was a unique feature of M1146 as described in a previous report on characterizing lipid compositions of M1146 (Thevenieau et al., 2014). A reason for such changes in BCFA profile is not reported. Deletions of the four gene clusters, ACT, RED, CPK, and CDA increase entire production of fatty acids, but would not be the main cause for increases in aiC15 concentrations. Synthesis of RED could be initiated by fabH, and some of BCAA catabolites could be

of

incorporated into a polyketide tail of RED, but anteiso-chains are minor (Mo et al., 2005). On the

ro

other hand, ACT and CPK are made up of acetyl-CoA and malonyl-CoA (Pawlik et al., 2007), and CDA is produced solely from amino acids (Kim et al., 2004). As a result, it may require

-p

more detailed metabolic flux balance analysis, or isotopomer labeled metabolic profiling to

re

provide some clues to explain increases of aiC15 concentrations.

At this point, we supplemented various amounts of leucine to cultures of M1146-FB to

lP

study whether there are improvements in cell growth and BCFA production as shown with SCOdA. The final concentrations of 1 ~ 5 mM leucine were supplemented at beginning of main

ur na

cultures. Leucine supplementations in M1146-FB strain somewhat decreased cell mass unlike in preliminary studies with SCOdA strain (Fig. S3A). BCFA production (Fig. S3B) were about the same throughout various concentrations. But due to decreases in cell mass, cell specific yield was increased by 25%, which was 26 mg/L/gcell, with 5 mM of leucine (Fig. S3C). Unfortunately,

Jo

leucine supplementation was not so effects on M1146-FB. Changes in FA profile and increases in BCFA production from M1146 strains agreed well with other reports on various BCFA producing species such as Bacillus subtilis. For example, leucine supplementation enhanced iC15 and iC16 production (Cybulski et al., 2002).

3.4 Blocking β–oxidation by deletion of fadD fadD as one of the most promising deletion targets as it initiates β-oxidation of FA catabolism. It was often modified and expressed to supply acyl-CoAs from FA degradations (Ford and Way, 2015). As a result, we constructed a fadD deletion mutant of M1146 strain (M1146dD) to stop degradation of our products, and maximize the FA titer. Cell mass of M1146dD strains with pIBR25::fabH (M1146dD-F), pIBR25::bkdABC

of

bkdABC (M1146dD-B), or pIBR25::fabH::bkdABC (M1146dD-FB) were reduced down to about 6

ro

~ 7 g/L (Fig. 5A). Despite the decreases in DCW of M1146dD strains, their abilities to produce FAs were highly enhanced. Total FA production in M1146dD was almost two times higher than

-p

that of M1146, total 259.2 mg/L with 228.1 mg/L of BCFA. M1146dD-B produced the most FAs,

re

392 mg/L of total FAs and 354.1 mg/L of BCFAs (Fig. 5B). It was 7.2 folds more than that of E. coli, and 1.3 folds more than that of previously reported highest titer (Bentley et al., 2016). Also,

lP

M1146dD-B did not require addition of expensive precursor, lipoic acid. Cell specific yield also increased to 64.4 mg/L/gcell in M1146dD-B, which was 11.8 fold increases compared to that of the

ur na

WT (Fig. 5C). These are the highest production titer and specific yield reported so far. fadD was also deleted in SCOdA (SCOdAD) to see if BCFA production could be increased without the losses in the total cell mass. SCOdAD carrying pIBR25::fabH (SCOdAD-F), pIBR25::bkdABC bkdABC (SCOdAD-B), or pIBR25::fabH::bkdABC (SCOdAD-FB) was

Jo

constructed. Losses in cell mass of SCOdAD-F, SCOdAD-B, and SCOdAD-FB were less severe than those of M1146dD strains (Fig. S4A), but unfortunately, they did not produce more FAs (Fig. S4B). All the major strains with their DCW and FAs production are listed in Table 3. One possible explanation to such losses in cell mass could be membrane structures of Streptomyces sp.. Lipid fractions of Streptomyces membranes are composed of about 25 % aiC15,

14 % iC16, and 40 % aiC17 (Zuzina et al., 1979). iC16 and aiC15 were the two most produced BCFAs in M1146 strains, and their production were increased by 3 to 4 fold of that of the WT. Such changes in BCFA profiles might have altered membrane structures of M1146 strains, causing reduction in some cell growth. Another possible explanation would be the roles of ACT in stress responses of S. coelicolor. Extracellular ACT is responsible for activation of soxR regulon, a regulator controlling a transport of small redox-active toxic molecules (Shin et al.,

of

2011). Exact functions of soxR in S. coelicolor is not well characterized (Cruz et al., 2010), but

ro

previous report on carbon-flux distribution of M1146 strain (Coze et al., 2013) explain potential

-p

limitations on cell growth due to oxidative stress.

re

3.5 Synthesis of BCFA from glucose

One of major metabolic pathways linked to fatty acid biosynthesis is glycolysis. Glycolysis is the

lP

major source of acetyl-CoA and malonyl-CoA, which become elongation units for FA biosynthesis. In addition, starter units of BCFAs can also be produced from glucose (Fig. 6A).

ur na

For that reason, production of SCFA in E. coli are greatly affected by glycolysis and expression levels of FAS genes (i.e. pyruvate dehydrogenase and fab genes) (Janβen and Steinbuchel, 2014). We overexpressed ilvBCD of BCAA synthesis pathway in SCOdA (SCOdA-I) and M1146 (M1146-I) to produce BCFAs from glucose. Because S. coelicolor can already produce the

Jo

starter units of BCFAs from BCAA, we also overexpressed poxB of glycolysis in SCOdA (SCOdA-P) and M1146 (M1146-P) to examine whether increasing supplies of acetyl-CoA and malonyl-CoA alone could also increase BCFA production. SCOdA-I and SCOdA-P exhibited about the same cell growth as the WT, but M1146-I and SCOdA-P had reduced cell mass (Fig. 6B). SCOdA-I produced about 125.7 mg/L of total FAs with

87.2 % BCFA (Fig. 6C). However, there were no differences in the FA production titers of M1146-I and M1146-P compared to that of M1146, which were 163.1 mg/L and 140.1 mg/L respectively (Fig. 6D).

4. Conclusion

of

In this work, 354.1 mg/L of BCFA, which was 90.3 % of the total FA, and cell specific FA yield of 64.4 mg/L/gcell were achieved by deletions of four major PKS gene clusters and fadD,

ro

along with overexpression of bkdABC. It was the highest titer ever reported from bacterial cultures. Deletions of PKS genes to redirect branched chain acyl-CoA fluxes towards FA

-p

synthesis were the most crucial factor in S. coelicolor. The same engineering strategies for PK

re

overproduction were also applicable in the synthesis of BCFA. Decreases in cell masses of S. coelicolor with increases in FA production were inevitable, but if cell growth could be optimized,

lP

it would further enhance the ability to produce BCFAs and their derivatives for next generation biofuels. Here we successfully developed and presented potentials of S. coelicolor as a cost

ur na

efficient host strain for BCFA production.

Acknowledgements

This research was supported by the National Research Foundation of Korea (NRF) funded by the

Jo

Ministry of Science, ICT & Future Planning (NRF-2017R1E1A1A01073523)

References

Arias, P., Fernandez-Moreno, M.A., Malpartida, F., 1999. Characterization of the pathwayspecific positive transcriptional regulator for actinorhodin biosynthesis in Streptomyces

coelicolor A3(2) as a DNA-binding protein. J. Bacteriol. 181, 6958-6968. Bart, J.C.J., Gucciardi, E., Cavallaro, S., 2012. Biolubricants: Science and Technology. Elsevier, Amsterdam, pp. 174-184. Bentley, G.J., Jiang, W., Guaman, L.P., Xiao, Y., Zhang, F., 2016. Engineering Escherichia coli to produce branched-chain fatty acids in high percentages. Metabo. Eng. 38, 148-158. Bergara, F., Ibarra, C., Iwamasa, J., Patarroyo, J.C., Aguilera, R., Márquez-Magaña, L.M., 2003.

of

CodY is a nutritional repressor of flagellar gene expression in Bacillus subtilis. J.

ro

Bacteriol. 185, 3118-3126.

Body, D.R., 1984. Branched-chain fatty acids. Handbook of Chromatography, CRC Press, Boca

-p

Raton, pp. 241-275.

re

Brakhage, A.A., 2004. Molecular biotechnology of fungal beta-lactam antibiotics and related peptide synthetases. Springer Sciences and Business Media, Germany, pp. 160-177.

lP

Brinsmade, S.R., Kleijn, R.J., Sauer, U., Sonenshein, A.L., 2010. Regulation of CodY activity through modulation of intracellular branched-chain amino acid pools. J. Bacteriol. 192,

ur na

6357-6368.

Choi, K.H., Heath, R.J., Rock, C.O., 2000. β-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) Is a Determining Factor in Branched-Chain Fatty Acid Biosynthesis. J. Bacteriol. 182, 365-370.

Jo

Cropp, T.A., Smogowicz, A.A., Hafner, E.W., Denoya, C.D., McArthur, H.A.I., Reynolds, K.A., 2000. Fatty-acid biosynthesis in a branched-chain α–ketoacid dehydrogenase mutant of Streptomyces avermitilis. Can. J. Microbiol. 46, 506-514.

Coze, F., Gilard, F., Tcherkez, G., Virolle, M.J., Guyonvarch, A., 2013. Carbon-flux distribution within Streptomyces coelicolor metabolism: a comparison between the actinorhodin-

producing strain M145 and its non-producing derivative M1146. PLoS One 8, e84151. Cruz, R.D., Gao, Y., Penumetcha, S., Sheplock, R., Weng, K., Chander, M., 2010. Expression of the Streptomyces coelicolor SoxR Reguln is Intimately Linked with Actinorhodin Production. J. Bacteriol. 192, 6428-6438. Cybulski, L.E., Albanesi, D., Mansilla, M.C., Altabe, S., Aguilar, P.S., Mendoza, D., 2002. Mechanism of membrane fluidity optimization: isothermal control of the Bacillus subtilis

of

acyl-lipid desaturase. Mol. Microbiol. 45, 1379-1388.

ro

Ford, T.J., Way, J.C., 2015. Enhancement of E. coli acyl-COA synthetase FadD activity on medium chain fatty acids. PeerJ 3, e1040.

-p

Gomez-Escribano, J.P., Bibb, M.J., 2010. Engineering Streptomyces coelicolor for heterologous

re

expression of secondary metabolite gene clusters. Microb. Biotechnol. 4, 207-215. Haushalter, R.W., Kim, W., Chavkin, T.A., The, L., Garber, M.E., Nhan, M., Adams, P.D.,

lP

Petzold, C.J., Katz, L., Keasling, J.D., 2014. Production of anteiso-branched fatty acids in Escherichia coli; next generation biofuels with improved cold-flow properties. Metab.

ur na

Eng. 26, 111-118.

Janβen, H.J., Steinbuchel, A., 2014. Fatty acid synthesis in Escherichia coli and its applications towards the production of fatty acid based biofuels. Biotechnol. Biofuels 7, 7. Jiang, W., Jiang, Y., Bentley, G.J., Liu, D., Xiao, Y., Zhang, F., 2015. Enhanced production of

Jo

branched-chain fatty acids by replacing β-ketoacyl-(acyl-carrier-protein) synthase III (FabH). Biotechnol. Bioeng. 112, 1613-1622.

Kaneda, T., 1991. Iso- and anteiso-fatty acids in bacteria: biosynthesis, function, and taxonomic significance. Microbiol. Rev. 55, 288-302. Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F., Hopwood, D.A., 2000. Practical Streptomyces

genetics. John Innes Foundation Norwich, Norwich, England. Kim, H.B., Smith, C.P., Micklefield, J., Mavituna, F., 2004. Metabolic flux analysis for calcium dependent antibiotic (CDA) production in Streptomyces coelicolor. Metab. Eng. 6, 313325. Kim, M., Yi, J.S., Kim, J., Kim, J.N., Kim, M.W., Kim, B.G., 2014. Reconstruction of a highquality metabolic model enables the identification of gene overexpression targets for

of

enhanced antibiotics production in Streptomyces coelicolor A3(2). Biotechnol. J. 9, 1185-

ro

1194.

Koppolu, V., Vasigala, V.K.R., 2016. Role of Escherichia coli in Biofuel Production. Microbiol.

-p

Insights. 9, 29-35.

re

Li, Y., Florova, G., Reynolds, K.A., 2005. Alteration of the fatty acid profile of Streptomyces coelicolor by replacement of the initiation enzyme 3-ketoacyl acyl carrier protein

lP

synthase III (FabH). J. Bacteriol. 187, 3795-3799.

McKenzie, N.L., Thaker, M., Koteva, K., Hughes, D.W., Wright, G.D., Nodwell, J.R., 2010.

ur na

Induction of antimicrobial activities in heterologous streptomycetes using alleles of the Streptomyces coelicolor gene absA1. J. Antibiot. (Tokyo) 63, 177-182. Mo, S.J., Kim, B.S., Reynolds, K.A., 2005. Production of Branched-Chain Alkylprodiginines in S. coelicolor by Replacement of the 3-Ketoacyl ACP Synthase III Initiation Enzyme,

Jo

RedP. Chem. Biol. 12, 191-200.

Okamoto, S., Lezhava, A., Hosaka, T., Okamoto-Hosoya, Y., Ochi, K., 2003. Enhanced expression of S-adenosylmethionine synthetase causes overproduction of actinorhodin in Streptomyces coelicolor A3(2). J. Bacteriol. 185, 601-609. Pawlik, K., Kotowska, M., Chater, K.F., Kuczek, K., Takano, E., 2007. A cryptic type I

polyketide synthase (cpk) gene cluster in Streptomyces coelicolor A3(2). Arch. Microbiol. 187, 87-99. Revill, W.P., Bibb, M.J., Hopwood, D.A., 1996. Relationships between fatty acid and polyketide synthases from Streptomyces coelicolor A3(2): characterization of the fatty acid synthase acyl carrier protein. J. Bacteriol. 178, 5660-5667. Shin, J.H., Singh, A.K., Cheon, D.J., Roe, J.H., 2011. Activation of the SoxR Regulon in

of

Streptomyces coelicolor by the Extracellular Form of the Pigmented Antibiotic

ro

Actinorhodin. J. Bacteriol. 193, 75-81.

J. Ind. Microbiol. Biotechnol. 27, 246-251.

-p

Smirnova, N., Reynolds, K.A. 2001. Branched-chain fatty acid biosynthesis in Escherichia coli.

re

Stirrett, K., Denoya, C., Westpheling, J., 2009. Branched-chain amino acid catabolism provides precursors for the Type II polyketide antibiotic, actinorhoin, via pathways that are

lP

nutrient dependent. J. Ind. Microbiol. Biotechnol. 36, 129-137. Swiatek, M.A., Gubbens, J., Bucca, G., Song, E., Yang, Y.H., Laing, E., Kim, B.G., Smith, C.P.,

ur na

van Wezel, G.P., 2013. The ROK family regulator Rok7B7 pleiotropically affects xylose utilization, carbon catabolite repression, and antibiotic production in Streptomyces coelicolor. J. Bacteriol. 195, 1236-1248. Tanaka, Y., Izawa, M., Hiraga, Y., Misaki, Y., Watanabe, T., Ochi, K., 2017. Metabolic

Jo

perturbation to enhance polyketide and nonribosomal peptide antibiotic production using triclosan and ribosome-targeting drugs. Appl. Microbiol. Biotechnol. 101, 4417-4431.

Thevenieau, F., Sambou, S., Virolle, M.J., Dulermo, T., 2014. Triacylglycerol-based Lipid Composition. Patent US14898926, AVRIL, Universite Paris-Sud. Yang, Y.H., Song, E., Kim, E.J., Le, K., Kim, W.S., Park, S.S., Hahn, J.S., Kim, B.G., 2009.

NdgR, an IclR-like regulator involved in amino-acid-dependent growth, quorum sensing, and antibiotic production in Streptomyces coelicolor. Appl. Microbiol. Biotechnol. 82, 501-511. Yao, L., Hammond, E.G., 2006. Isolation and melting properties of branched chain esters from lanolin. J. Am. Oil Chem. Soc. 83, 547-552. Yi, J.S., Kim, M., Kim, E.J., Kim, B.G., 2018. Production of pikromycin using branched chain

of

amino acid catabolism in Streptomyces venezuelae ATCC 15439. J. Ind. Microbiol.

ro

Biotechnol. 45, 293-303.

Yi, J.S., Kim, M.W., Kim, M., Jeong, Y., Kim, E.J., Cho, B.K., Kim, B.G., 2017. A Novel

-p

Approach for Gene Expression Optimization through Native Promoter and 5’ UTR

ACS Synth. Biol. 6, 555-565.

re

Combinations Based on RNA-seq, Bibo-seq, and TSS-seq of Streptomyces coelicolor.

lP

Xu, K., Lv, B., Huo, Y.X., Li, C., 2017. Toward the lowest energy consumption and emission in biofuel production: combination of ideal reactors and robust hosts. Curr. Opin. Biotechnol.

ur na

50, 19-24.

Zhang, W., Ferreira, J.P., Tang, Y., 2009. The Metabolic Pathway Engineering Handbook: Fundamentals. CRC Press, Florida. Zhang, Z.C., Dery, M., Zhang, S., Steichen, D., 2004. New process for the production of

Jo

branched-chain fatty acids. J. Surfact. Deterg. 7, 211-215.

Zuzina, M.L., Efimova, T.P., Tereshin, I.M., 1979. Composition of Membranes and Mycelia of Streptomyces levoris at Different Stages of Growth. Curr. Microbiol. 2, 215-217.

Tables Table 1. List of plasmids and cosmids used in this study Strains or Plasmids

Descriptions

References

Plamids

pdCos6196 pIBR25 pIBR25::bkdABC pIBR25::fabH

Streptomyces plasmid containing cre recombinase gene

Swiatek et al. 2013

actII-orf4 deletetion cassette cloned into EcoRI/XbaI site of pSupercos1

this study

fadD deletion cassette inserted and reaplced fadD of St2G5.17 cosmid

this study

of

pSupCos5085

Yang et al. 2009

ro

pUWLCRE

A template vector for amplifications of gene deletion cassettes

Streptomyces vector with SCP2* origin; AmpR and tsr marker bkdABC cloned into XbaI/EcoRI sites of pIBR25

-p

pIJ773

fabH cloned into PstI/XbaI sites of pIBR25

Yi et al. 2017

Kim et al. 2014 Yi et al. 2018

pIBR25 containing fabH (PstI/XbaI) and bkdABC (XbaI/EcoRI)

this study

pIBR25::ilvBCD

ilvBCD cloned into XbaI/HindIII sites of pIBR25

this study

poxB cloned into BamHI/PstI sites of pIBR25

this study

a S. coelicolor cosmid containing fadD

this study

Cosmids

Jo

ur na

St2G5.17

lP

pIBR25::poxB

re

pIBR25::fabH::bkdABC

Table 2. List of bacterial strains used in this study Strains or Plasmids

Descriptions

References

Streptomyces strains Streptomyces coelicolor A3 (2) M145

Wild type (WT) strain

Yi et al. 2017

S. coelicolor A3 (2) M145 with ΔactII-orf4

this study

SCOdAD

S. coelicolor A3 (2) M145 with ΔactII-orf4 and ΔfadD

this study

SCOdAD-F

SCOdAD containing pIBR25::fabH

this study

SCOdAD-B

SCOdAD containing pIBR25::bkdABC

this study

SCOdAD-FB

SCOdAD containing pIBR25::fabH::bkdABC

this study

SCOdA-I

SCOdA containing pIBR25::ilvBCD

SCOdA-P

SCOdA containing pIBR25::poxB

this study this study

ro

M1146

of

SCOdA

S. coelicolor A3 (2) M145 with ΔactΔredΔcpkΔcda

Coze et al. 2013

M1146 containing pIBR25::fabH

M1146-B

M1146 containing pIBR25::bkdABC

M1146-FB

M1146 containing pIBR25::fabH::bkdABC

this study

M1146 strain with ΔfadD

this study

re

M1146dD

-p

M1146-F

this study this study

M1146dD containing pIBR25::fabH

this study

M1146dD-B

M1146dD containing pIBR25::bkdABC

this study

M1146dD-FB

M1146dD containing pIBR25::fabH::bkdABC

this study

lP

M1146dD-F

M1146 containing pIBR25::ilvBCD

this study

M1146-P

M1146 containing pIBR25::poxB

this study

ur na

M1146-I

E. coli strains

DH5α

a host for gene constructions

JM110

a strain for demethylation of plasmids

Jo

ET12567

a strain for conjugations of S. coelicolor cosmids

ThermoFisher Stratagene ATCC

Table 3. List of S. coelicolor strains and their FA production fabH

BCFA (%)

BCFA (mg/L)

Growth (g/L)

Cell Specific Yield (mg/L/gcell)

53.1

58.1

30.9

11.7

4.5

72.0

60.2

43.3

9.9

7.3

184.5

90.2

166.4

12.9

14.3

170.5

89.8

153.1

9.6

17.8

+

154.0

87.1

134.1

6.2

24.9

+

233.3

86.9

202.7

10.4

22.4

259.2

88.0

228.1

8.0

32.4

191.4

85.1

162.9

6.2

31.0

+

392.1

90.3

354.1

6.3

62.1

+

219.7

84.9

186.6

8.0

27.3

bkdABC

WT SCOdA

-

M1146

-

-

M1146-F

-

-

M1146-B

-

-

M1146-FB

-

-

M1146dD

-

-

-

M1146dD-F

-

-

-

M1146dD-B

-

-

-

M1146dD-FB

-

-

-

+

+

+

Jo

ur na

lP

re

-p

+

of

fadD

Total FA (mg/L)

ro

actII-orf4

act/red/ cda/cpk

Figure legends Figure 1. Overall schemes of engineering and a BCAA degradation pathway of Streptomyces coelicolor, leading to synthesis of FAs and secondary metabolites. Overexpression targets are indicated by bold arrows, and deletion targets are marked by bold X.

of

Figure 2. (a) Total FA production and (b) growth curves from the WT, SCOdA, and SCOdA with BCAA supplementation. (c) FA contents of the WT and SCOdA, and (d) SCOdA with BCAA

ro

supplementation. BCAAs were supplemented with final 2 mM concentrations. Deletion of actII-

-p

orf4 improved FA production by increasing iC16 content in SCOdA.

re

Figure 3. (a) Growth curves, (b) FA production, and (c) FA content profiles of the WT S.

lP

coelicolor, SCOdA, and M1146.

Figure 4. (a) Growth curves, (b) FA production, and (c) cell specific yield of the M1146 strains

ur na

overexpressing fabH and bkdABC. Due to losses in cell mass, fabH and bkdABC single overexpression have about the same production titer as that of M1146. The two genes together

Jo

have synergic effects on the FA production, resulting notable increase in the production titer.

Figure 5. (a) Growth curves, (b) FA production, and (c) cell specific yield of the M1146dD strains. Losses in cell mass were more severe in ΔfadD mutants. But the highest cell specific yield was doubled in M1146dD-B.

Figure 6. (a) Glucose and BCAA metabolism leading to synthesis of FA precursors. ilvBCD and

poxB are promising overexpression candidates for increasing BCFA production. (b) Growth curves and FA production from (c) SCOdA and (d) M1146 strains overexpressing ilvBCD and

Jo

ur na

lP

re

-p

ro

of

poxB.

re

lP

ur na

Jo

Figure 1.

-p

ro

of

of

ro

-p

re

lP

ur na

Jo Figure 2.

of

ro

-p

re

lP

ur na

Jo Figure 3.

Figure 4.

of

ro

-p

re

lP

ur na

Jo

Figure 5.

of

ro

-p

re

lP

ur na

Jo

Figure 6.

of

ro

-p

re

lP

ur na

Jo