Exploiting members of the BAHD acyltransferase

family to synthesize multiple hydroxycinnamate

and benzoate conjugates in yeast

Outcomes• The synthesis of valuable products such as rosmarinic acid, chlorogenic acid, glycerol, polyamine, monolignol, malate and fatty

alcohol hydroxycinnamates was achieved for the first time in yeast.

Aymerick Eudes et al.(2016). “Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast”. Microb. Cell Fact. doi: 10.1186/s12934-016-0593-5

Background• The production of renewable

chemicals using engineered

microbes is an alternative route

to energy-intensive chemical

syntheses that consume

petroleum-based precursors.

• Identification of appropriate

enzymes is a prerequisite for

the design and implementation

of metabolic pathways in

microbes and bioenergy crops.

Approach• BAHD acyltransferases that use

hydroxycinnamoyl-CoA lignin

precursors and/or benzoyl-CoA

as donors were characterized

for the synthesis of valuable

metabolites in S. cerevisiae.

Significance• The BAHD acyltransferases characterized in the work can be expressed in bioenergy crops for the

production of co-products and to add value to plant biomass.

1) Mechanism of acylation catalyzed by BAHD acyltransferases. BAHD acyltransferases using 4-hydroxycinnamoyl-CoAs (R = 4-hydroxystyrene) and benzoyl-CoAs (R = benzene) as donors were used for this study. R’ = BAHD acceptor.

Hydroxycinnamoyl-CoA:glyceroltransferase

Hydroxycinnamoyl-CoA:spermidinetransferase

Hydroxycinnamoyl-CoA / benzoyl-CoA:alcohol transferase

Hydroxycinnamoyl-CoA:fatty alcoholtransferase

Hydroxycinnamoyl-CoA:monolignoltransferase

Rosmarinic acid synthase Hydroxycinnamoyl-CoA:Quinatetransferase

+BAHD

CoA-activated

acyl donorsAcyl acceptors

Ester bond

Amide bond

Cinnamates

BAHDs

Acceptor

Cinnamoyl-CoAs + Acceptor

Supplied Produced

4CL

Cinnamoylconjugates

2) Strategy for the synthesis of cinnamate and benzoate conjugates. Yeast strains expressing various BAHDs are fed with cinnamate or benzoate donors in combination with adequate acceptor molecules. The produced conjugates are detected from the culture medium. 4CL, 4-coumarate:CoA ligase.

3) Name of the BAHDs synthesized for expression in yeast and examples of hydroxycinnamate and benzoate conjugates produced in this work.

Hydroxycinnamoyl-CoA:malatetransferase

Loss of inositol phosphorylceramide sphingolipid

mannosylation induces plant immune responses

and reduces cellulose content in Arabidopsis

Outcomes• GMT1 mannosylates a class of sphingolipids (GIPCs) which

are abundant in the plant plasma membrane (Figure 1).

• Disruption of this glycosylation in gmt1 causes a specific

decrease in the plasma-membrane synthesized cellulose.

Golgi-synthesized polysaccharides, including pectin, were

unaffected.

• gmt1 plants also have a constitutively active defense response.

Fang et al. (2017). "Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses

and Reduces Cellulose content in Arabidopsis". Plant Cell. doi, 10.1105/tpc.16.00186

Background• Plant Golgi-localized glycosyltransferases (GTs) are involved in

non-cellulosic polysaccharide biosynthesis, as well as lipid and

protein glycosylation.

• Previous work had suggested that GMT1, from CAZy family

GT64, was Golgi localized and involved in pectin biosynthesis,

but its function was unknown.

Significance• These results describes the function of the first plant GT64,

and only the second GT involved in GIPC biosynthesis. It

uncovers a new potential role for GIPCs in regulating cellulose

content. The mechanism by which this could occur is unknown

and will be explored further to applications in bioenergy.

Approach• Here, we fully characterized mutants in GMT1, as well as

heterologously expressed the protein in yeast and tobacco.Above: GIPCs, a class of sphingolipid, are an abundant component of the outer leaflet of the plant plasma membrane. Loss of a single mannose on the GIPC headgroup has a major impact on plant development. Below: 13C CP-MAS solid state NMR (left) and the subtraction spectra (WT-gmt1; right) reveals that cellulose signals are specifically lost in the gmt1 stem (labelled C1-C6, where C1 is the anomeric carbon of glucose in cellulose).

Enrichment of the Plant Cytosolic Fraction

Outcomes• A detailed protocol for enriching the cytosolic fraction from

cell cultures developed at JBEI.

• The method highlights the necessity for protoplasting to

ensure high levels of purity

Lao et al. (2017). “Enrichment of the Plant Cytosolic Fraction”. In N. L. Taylor & A. H. Millar (Eds.), Isolation of Plant

Organelles and Structures: Methods and Protocols (pp. 213-232). New York, NY: Springer New York.

Background• The cytosol is at the core of cellular metabolism and

contains many important metabolic pathways, such as

glycolysis, gluconeogenesis and the pentose phosphate

pathway. Despite the importance of this matrix, few

attempts have sought to specifically enrich thiscompartment from plants.

Significance• The approach yields samples suitable to define the

proteome of the cytosol, a much understudied fraction

within the plant cell. The approach is high suitable for

comparative proteomic approaches.

1) Schematic outlining the enrichment of the cytosolic fraction

from rice cells using a protoplast rupture method.

Approach• We outline a detailed protocol for the enrichment of the

cytosolic fraction from rice cell cultures. The approach

highlights the requirement for protoplasting cells to

ensure reduced contamination from organelles. This

approach results in a highly enriched fraction and

expands work conducted at JBEI focussing on

Arabidopsis cytosolic fractions.

2) Immunoblotting with organelle marker antibodies demonstration

the removal of contaminating organelles by centrifugation.

Structure and mechanism of NOV1, a

resveratrol-cleaving dioxygenase

Outcomes• The structure of NOV1 was determined in complex with a

representative substrate (resveratrol), a representative product

(vanillin), and without ligand bound.

• These structures, along with EPR analysis, allow us to propose a

mechanism in which a ferric-superoxide reacts with substrate

activated by deprotonation of a phenol group at position 4 of the

substrate.

McAndrew et al. (2016) ”Structure and mechanism of NOV1, a resveratrol-cleaving

dioxygenase”. Proc Natl Acad Sci U S A. DOI: 10.1073/pnas.1608917113

Background• NOV1 is a stilbene cleavage oxygenase (SCO). SCOs cleave the

central double bond of stilbenes, forming two phenolic aldehydes.

Stilbenes, such as resveratrol, are produced by plants and they

are also formed from lignin during kraft pulping.

• SCOs are related to carotenoid cleavage oxygenases (CCOs),

which cleave β-carotene or apocarotenoids. Carotenoids play

important roles in photosynthesis and light perception in the eye.

Significance• The conversion of lignin, which accounts for ∼30% of plant cell

wall carbon, into chemicals or fuels could have a significant

impact on the economics of processing lignocellulosic biomass.

Ultimately, enzymes like NOV1 could assist in the biological

valorization of dimeric fragments derived from lignin and so

contribute to the sustainable operation of a biorefinery for the

production of biofuels and other bioproducts.

Approach• We used X-ray crystallography to determine the first structure of

an SCO. EPR analysis was also used to help determine the

enzyme mechanism.

• Collaboration with Brian Fox @ GLBRC

Overall Structure of NOV1. (A) NOV1 is a seven-bladed β-propeller. An

iron coordinated by four histidines is located down the central axis. Dioxygen

binds to the iron, and resveratrol binds with its central double bond proximal

to the oxygen. (B) A surface slice representation, oriented perpendicular to

the β-propeller axis shows the shape of the active site cavity.

A B

Mechanism of NOV1. (A) Formation of ternary complex of Fe(III)-

superoxo NOV1, O2 and alkene substrate, and contributions of

deprotonation of 4′-OH by Y101 and K135 in activation of the substrate. (B)

Intermediate formed prior to formation of a C–O bond. (C) Intermediate

formed after formation of the first C–O bond. (D) Cleavage of the O–O

bond and formation of the second C–O bond. (E) Cleavage of the C–C

bond and restoration of the Fe(II) enzyme. (F) Reaction products.

A B C

F E D

Sequential enzymatic saccharification and

fermentation of ionic liquid and organosolv

pretreated agave bagasse for ethanol production

Outcomes• IL pretreatment reduced lignin by 28% and xylan by 50%

while OV solubilizes 86% xylan and 45% of lignin.

• High glucan (>90 %) and xylan (>83 %) conversion was

obtained with both pretreated samples that lasted 18h.

• During the fermentation stage (48 h), 12.1 and 12.7 kg of

ethanol were produced per 100 kg of untreated AGB for

IL and OV, respectively.

A) Mass balance per 100 kg of

untreated AGB during sequential

enzymatic saccharification and

fermentation (SESF) for IL and

OV pretreatments. When the Agave productivity is

considered, 3067-7082 L/ha year is

obtained, favorable when compared to

corn (2050 L/ha year) or sugarcane

(4900 L/ha year).

Pérez-Pimienta et al. (2016). “Sequential enzymatic saccharification and fermentation of ionic liquid and organosolv

pretreated agave bagasse for ethanol production”. Bioresource Technology (2017) 2225, pp. 191-198.

Background• Agave bagasse (AGB) has gained recognition

as a drought-tolerant biofuel feedstock with

high productivity in semiarid regions.

• Studies on AGB for ethanol production have

used only the separate hydrolysis and

fermentation (SHF) strategy.

Significance• These comparative analyses showed the advantages of SESF using IL

and OV in a biorefinery configuration where a better understanding of AGB

recalcitrance is key for future applications.

B) XRD spectra of untreated and

pretreated AGB.

1) The crystallinity index (CrI) of

untreated AGB was 39.0% whereas IL

decreased to 22.6% and OV increased

to 44.6%. 2) Spectrum of untreated

AGB shows distinctive peaks at 2θ =

14.8 °, 24.2 °, 30.0 ° and 38.0 °from calcium oxalate.

Approach• Comparative analysis of ionic liquid (IL) and

organosolv (OV) pretreatment technologies in

AGB was performed using a sequential enzymatic

saccharification and fermentation (SESF) strategy

with cellulolytic enzymes and metabolic

engineered Escherichia coli strain MS04.

A

B

C) Scanning electron (top) and confocal fluorescence

(bottom) of untreated and pretreated AGB.

Intact plant cell wall structure of AGB is shown while

pretreatment with [C2C1Im][OAc] in AGB resulted in a structural

modification into the organization of macrofibrils with a rough

and swollen surface.

C

Nitrogen amendment of green waste impacts

microbial community, enzyme secretion and

potential for lignocellulose decomposition

Outcomes• Carbon/nitrogen ratios of 25–30 are considered ideal for thermophilic biological

deconstruction.

• Carbon/nitrogen ratios of the green waste after nitrogen amendment ranged between 25 and

32, however, even within this range, significant shifts in the microbial community, enzyme

production and potential for lignocellulose hydrolysis were observed.

Yu et al. (2016). "Nitrogen amendment of green waste impacts microbial community, enzyme secretion and potential

for lignocellulose decomposition". Process Biochemistry. doi, 10.1016/j.procbio.2016.11.002

Background• Each year, 167 million metric tons of municipal

solid wastes are sent to landfills in the U.S and

about 44.9% of these wastes are organic

• Microorganisms involved in biomass

deconstruction are an important resource for

organic waste recycling and enzymes for

lignocellulose bioconversion.

Significance• The results suggest nitrogen levels present in green waste bioconversion processes could

be better tuned to achieve more efficient deconstruction of recalcitrant polysaccharides and

discovery of enzymes for bioconversion.

Approach• The goals of this study were to elucidate the

biological mecha-nisms of enhanced green

waste decomposition with nitrogen amendment

through examination of xylanase and

endoglucanase secretion, microbial community

restructuring and potential for green waste

deconstruction.

Mean relative abundance (%) of phyla in bacterial

communities and classes in fungal communities

Ionic liquid-tolerant microorganisms and microbial

communities for lignocellulose conversion to

bioproducts

Outcomes• IL tolerant microorganisms from environmental

samples by generating less complex lignocellulolytic

microbial communities and facilitating the discovery

of potential enzymes and microorganisms for

biomass deconstruction.

Yu at al. (2016). "Ionic liquid-tolerant microorganisms and microbial communities for lignocellulose conversion to

bioproducts". Appl Microbiol Biotechnol, 100(24), 10237-10249. doi, 10.1007/s00253-016-7955-0

Background• While effective at pretreatment, certain ionic liquids

inhibit not only the enzyme activities but also the

growth and productivity of microorganisms used in

downstream hydrolysis and fermentation processes

• The discovery of IL tolerance in environmental

microbial communities and individual microbes has

lead to the proposal of molecular mechanisms of

resistance.

Significance• A combined approach that includes ILs designed for

reduced toxicity and robust IL-tolerant

microorganisms will ultimately result in more efficient

and economical IL-pretreatment based bioconversion

processes.

Approach• This is a review paper on recent progress on

discovering IL-tolerant microorganisms, identifying

metabolic pathways and mechanisms of tolerance,

and engineering microorganisms for IL tolerance

Examples of ionic liquid tolerance organisms

Bio-based production of fuels and industrial

chemicals by repurposing modular polyketide

synthases: opportunities and challenges

Yuzawa, et al. (2016). “Bio-based production of fuels and industrial chemicals by repurposing antibiotic-producing type I

modular polyketide synthases: challenges and opportunities” J Antibiot (Tokyo) doi: 10.1038/ja.2016.136.

Background• Complex polyketides comprise a large number of

natural products that have broad application in

medicine and agriculture.

• They are produced in bacteria and fungi from enzyme

complexes named type I modular polyketide synthases

(PKSs) that contain discrete enzymatic domains

organized into modules.

• The modular nature of PKSs has enabled a multitude

of efforts to “repurpose” the PKS genes to produce

fuels and industrial chemicals in a predicted manner.

Significance• These results greatly enhance the mechanistic

understanding of PKS and pave the way for

exploitation of PKS as a platform to produce fuels

and industrial chemicals.

(C)

AT

LipPks1+TE

ACP KS AT KR ACP TE

R

O

S

(A)

Load Module 1

AT

AT-swapped LipPks1+TE

ACP KS KR ACP TE

R

O

S

Load Module 1

AT

S

O

R

HO

R

O

OH

OH

AT KS ATACP KR ACP TEER

O

HO

O

OH

S

O

O

HOO

HO

O

S

DH

KR-swapped BorA2+TE

Module 1

BorA1

Load

AT

KR-inactivated LipPks1+TE

ACP KS AT KR ACP TE

R

O

S

(B)

Load Module 1

AT

KR-inactivated, AT-swapped LipPks1+TE

ACP KS KR ACP TE

R

O

S

Load Module 1

AT

S

O

R

O

R

O

R

O

R =

Starter acyl-CoA = Propionyl n-Butyryl Isobutyryl 2-Methylbutyryl Isovaleryl

R =

Starter acyl-CoA = Propionyl n-Butyryl Isobutyryl 2-Methylbutyryl Isovaleryl

R

O

OH

OH

S

O

R

HO

S

O

R

O

Adipic acid

Opportunities• We have repurposed PKSs to produce 3-hydroxy

acids, ketones, and diacids that could have

applications as fuels or industrial chemicals.

Challenges• The challenge is to make these compounds at

reasonable cost. We need to find the ‘best’ host for

production of type I modular PKS-based fuels and

industrial chemicals.

Enrichment of Golgi Membranes from Triticum

aestivum (Wheat) Seedlings

Outcomes• A detailed protocol for enriching Golgi membranes from

plant material (seedlings) using approaches developed at

JBEI

• The method results in an enrichment rather than an

organelle purification

Zeng et al. (2017). “Enrichment of Golgi Membranes from Triticum aestivum (Wheat) Seedlings”. In N. L. Taylor & A. H. Millar

(Eds.), Isolation of Plant Organelles and Structures: Methods and Protocols (pp. 131-150). New York, NY: Springer New York.

Background• The complex collection of membrane structures

comprising the Golgi apparatus has historically been

difficult to purify from plant material. Density

centrifugation has typically been used to enrich Golgi

membranes from microsomal preparations, and aside

from minor adaptations, the approach is still widelyemployed by the field.

Significance• The approach yields an adequate enrichment of Golgi

membrane to enable a variety of biochemical analyses.

Knowledge of the published Golgi proteome can be

leveraged to enable proteomic surveys of resultant

samples.

1) Schematic outlining the enrichment of Golgi membranes from

wheat by density centrifugation

Approach• We outline a detailed protocol that we have extensively

refined for the enrichment of Golgi membranes from

wheat seedlings. While the approach results in a

relatively impure preparation, it is suitable for downstream

processes such as comparative proteomic or biochemical

assays.

2) Validation of the Golgi enrichment process using

immunoblotting with organelle marker antibodies and enzyme

assays (UDPase).

Significance• We provide the largest, most comprehensive and well-characterized toolkit for S. cerevisiae strain

engineering to date, allowing for quick and easy metabolic pathway construction.

• Using our tools, we improve taxadiene titers in S. cerevisiae to the highest levels reported to date.

Providing better Cas9-based tools for genetic

engineering in Saccharomyces cerevisiae

Background• Strain development remains slow and

laborious because of difficulties anticipating the

combined effect of different expression parts

and conditions in S. cerevisiae.

Approach• We developed a Cas9-based toolkit to quickly

institute genetic changes in S. cerevisiae for

optimizing heterologous gene expression. C

B

AI II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI

106308

RDS1

720CAN1

511

SAP155

607 1014

911

805 1206

1114

1021

HIS3

1309

1414

1622

YOLCdelta1

YPRCdelta15

208

416

C

PTEF1

GFPTADH1

upX dnX

Constructed many high-efficiency Cas9-

sgRNA plasmids for easy integration

A diversity context library on taxadiene

synthase, using several of our parts, resulted

in a 25-fold improvement in taxadiene titer

Outcomes• Constructed high-efficiency, Cas9-sgRNA

plasmids targeting 23 characterized integration

loci.

• Characterized 37 standardized promoters in

different grow phases and media

• Validated functions for 10 protein tags conferring

specific protein localization, turnover or

solubility.

• Provide a software tool to allow for easy utilization

of parts.

A thorough single-cell fluorescent

evaluation of promoters across time and

media discovered a range of activation

profiles including stationary phase and

exponential phase induction.

Reider Apel et al. (2016). “A Cas9-based toolkit to program gene expression in

Saccharomyces cerevisiae”. Nucleic Acid Research, doi: 10.1093/nar/gkw1023.