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ABSTRACT Plants represent a safe and cost-effective platform for producing high-value proteins with pharmaceutical properties; however, the ability to accumulate these in commercially viable

quantities is challenging. Ideal crops to serve as biofactories would include low-input, fast-growing, high-biomass species such as sugarcane. The objective of this study was to develop an

efficient expression system to enable large-scale production of high-value recombinant proteins in sugarcane culms. Bovine lysozyme (BvLz) is a potent broad-spectrum antimicrobial enzyme

used in the food, cosmetics and agricultural industries. Here, we report a novel strategy to achieve high-level expression of recombinant proteins using a combinatorial stacked promoter

system. We demonstrate this by co-expressing _BvLz_ under the control of multiple constitutive and culm-regulated promoters on separate expression vectors and combinatorial plant

transformation. BvLz accumulation reached 1.4% of total soluble protein (TSP) (10.0 mg BvLz/kg culm mass) in stacked multiple promoter:_BvLz_ lines, compared to 0.07% of TSP (0.56 mg/kg) in

single promoter:_BvLz_ lines. BvLz accumulation was further boosted to 11.5% of TSP (82.5 mg/kg) through event stacking by re-transforming the stacked promoter:_BvLz_ lines with additional

_BvLz_ expression vectors. The protein accumulation achieved with the combinatorial promoter stacking expression system was stable in multiple vegetative propagations, demonstrating the

feasibility of using sugarcane as a biofactory for producing high-value proteins and bioproducts. SIMILAR CONTENT BEING VIEWED BY OTHERS IDENTIFICATION OF A SUGARCANE BACILLIFORM VIRUS

PROMOTER THAT IS ACTIVATED BY DROUGHT STRESS IN PLANTS Article Open access 26 March 2024 POPLAR TRANSFORMATION WITH VARIABLE EXPLANT SOURCES TO MAXIMIZE TRANSFORMATION EFFICIENCY Article

Open access 08 January 2025 ENERGY-EFFICIENT PRODUCTION OF VACCINE PROTEIN AGAINST PORCINE EDEMA DISEASE FROM TRANSGENIC LETTUCE (_LACTUCA SATIVA_ L.) Article Open access 24 September 2022

INTRODUCTION Recombinant proteins are currently being produced in cultured cell-based systems in mammals, microbes (bacteria and yeast), insects and plants, as well as in transgenic animals

(reviewed by Demain and Vaishnav)1. Transgenic plants constitute an attractive system for expression and production of a variety of proteins and biomolecules due to their efficient

eukaryotic protein synthesis, high scalability, relatively low production costs and environmental footprint2,3,4. However, selecting suitable hosts and expression vectors are key

considerations since protein accumulation is determined by expression levels. Important factors to consider when selecting a plant-based production platform include biomass yield per

hectare, recombinant protein yield per unit biomass, ease of transformation, scalability and safety5. Sugarcane (_Saccharum_ spp. hybrids), a key feedstock in the expanding bioeconomy as a

sugar and bioenergy crop6, is an ideal platform for recombinant protein production for several reasons: (1) It is a relatively fast growing tropical grass with the highly efficient C4

photosynthetic pathway, conferring high biomass production capacity with yields of up to 41.3 tons of biomass (harvested dry mass) per hectare per annum7,8; (2) it is highly efficient in

utilizing radiation, water and nutrients to produce a large biomass and hence a higher recombinant protein yield; (3) it is readily amenable to genetic engineering, with established

transformation and tissue regeneration techniques9,10; and (4) it has a low risk of out-crossing recombinant genes due to its primarily vegetative means of propagation; natural reproductive

propagation in many temperate and subtropical regions is rare due to its photoperiod sensitivity. Sugarcane was used as biofactory for the production of new biomolecules such as

bioplastics11,12,13,14,15, alternative sugars (sorbitol and isomaltulose)16,17,18, and recombinant proteins including the human cytokine granulocyte macrophage colony stimulating factor

GM-CSF19, canecystatins (cysteine protease inhibitors) CaneCP-1, CaneCP-2 and CaneCP-320,21,22, and the cellulolytic enzymes, endoglucanase and cellobiohydrolases I and II23,24. Accumulation

levels of these recombinant proteins ranged from 0.02 to 2.0% of total soluble protein (TSP) in leaves. However, very few attempts have so far been made to express recombinant proteins in

sugarcane culms (reporter proteins)25, which constitute the largest fraction of harvestable biomass and would be an ideal platform for production of bulk proteins. Bovine lysozyme (BvLz) is

more important industrially than other lysozymes because of its potent broad-spectrum antimicrobial activity26,27, especially against Gram-negative bacteria and fungi at concentrations as

low as 25 ppm, its sixfold higher chitinase activity than that of chicken lysozyme28, and its thermal stability and resistance to proteolysis29. BvLz, unlike other enzymes, possesses

biochemical properties that make it suitable for protein extraction and purification, such as stability over a broad pH range, thermal stability, resistance to proteolysis and convenient

quantification assays30,31. In this study, we demonstrate the feasibility of developing sugarcane as an expression platform for production and purification of recombinant proteins at high

levels, i.e. up to 11.5% of TSP (82.5 mg protein/kg culm mass). Multiple promoters (constitutive or culm-regulated) on separate expression vectors were stacked by combinatorial plant

transformation approach to boost production levels of recombinant _bovine lysozyme_ (_BvLz_), which was codon-optimized for expression in monocots. A double terminator or 3′ untranslated

region (UTR) was incorporated for improved transcript stability. Enzymatic activity and enzyme-linked immunosorbent assays (ELISA) of _BvLz_ transgenic sugarcane culm protein extracts and

clarified juice confirmed the presence of an intact and fully active BvLz enzyme, which accumulated in multiple vegetative generations at levels as high as 10.0 mg/kg (1.4% of TSP) in lines

co-expressing _BvLz_ from stacks of three or four different promoters on separate vectors, compared to 0.56 mg/kg (0.07% of TSP) in lines expressing _BvLz_ from a single promoter vector. We

further observed BvLz accumulation up to 82.5 mg/kg (11.5% of TSP) through event stacking by re-transforming the stacked promoter:_BvLz_ transgenic lines with additional _BvLz_ expression

vectors. RESULTS AND DISCUSSION THE COMBINATORIAL PROMOTER AND EVENT STACKING RESULT IN INCREASED RECOMBINANT PROTEIN PRODUCTION IN TRANSGENIC SUGARCANE CULMS A salient feature of

combinatorial transformation, a special case of co-transformation32, is that there is no theoretical limit to the number of expression vectors that can be co-transformed. To enable

high-levels of recombinant protein production in sugarcane culms, we developed a combinatorial promoter and event stacking system and demonstrated its application in producing a high-value

bovine lysozyme (BvLz) protein. This was facilitated by the availability of a set of constitutive and culm-regulated promoters previously isolated from sugarcane, in addition to the common

maize _ubiquitin 1_ promoter (pUbi)33. These include the culm-regulated promoters for _Sugarcane bacilliform virus_ (pSCBV21)34 and sugarcane _dirigent16_ (pSHDIR16) gene35, and the

constitutive promoters for sugarcane _proline-rich protein_ (pSHPRP)36 and _elongation factor 1α_ (pSHEF1α)36 genes. Furthermore, conditions for small-scale and large-scale extraction and

clarification of recombinant BvLz from sugarcane culm extracts and juice were optimized at our Pilot Plant and BioSeparation Facilities30,37. The essential design of the resulting new

combinatorial promoter stacking system is illustrated in Fig. 1. The system consisted of co-expressing the codon-optimized _BvLz__m_, from a stack of multiple promoters on separate

expression vectors in sugarcane by combinatorial transformation. A double terminator, composed of the _Cauliflower mosaic virus_ (CaMV) 35S terminator (35ST) and the _Agrobacterium

tumefaciens_ nopaline synthase terminator (NOST), or the 3′UTR of _Sorghum mosaic virus_ (SrMV), was fused to the coding region of _BvLz__m_ to enhance transcript stability38,39 (Fig. 1). To

test the stacking promoter gene expression system, embryogenic calli (2 month-old) and leaf roll discs (12 day-old), prepared from several commercial sugarcane varieties were co-transformed

biolistically with the multiple promoter:_BvLz__m_ expression vectors, using the _bar_ gene (phosphinothricin acetyl transferase) as a selectable marker. Several independent transgenic

_BvLz__m_ lines, identified by Southern blot analysis (Fig. 2a; Supplementary Fig. S2), were generated from the combinatorial transformation of sugarcane with single, double, triple or

quadruple promoter:_BvLz__m_ expression vectors (Table 1). These represent: (1) 43 lines (114 plants) expressing _BvLz__m_ from a single promoter, (2) 10 lines (52 plants) expressing

_BvLz__m_ from a double promoter stack, (3) 24 lines (318 plants) expressing _BvLz__m_ from a triple promoter stack, and (4) 23 lines (76 plants) expressing _BvLz__m_ from a quadruple

promoter stack (Table 1). The integration and size of each respective _BvLz__m_ expression vector (promoter, _BvLz__m_, terminator and/or 3′UTR) in the single and stacked multiple

promoter:_BvLz__m_ lines were confirmed by Southern blot hybridization with a full-length _BvLz__m_ probe (Fig. 2a; Supplementary Fig. S2) and by PCR using primers encompassing each of the

different promoter:_BvLz__m_-terminator cassettes (Fig. 3; Supplementary Fig. S4, S5 and S6). All lines were analyzed for their _BvLz__m_ transcript levels by northern blot hybridization

(Fig. 2b; Supplementary Fig. S3) as well as for their BvLzm accumulation by ELISA (Table 1; Fig. 2c for ELISA). For representative lines, yield was also determined by an enzyme activity

assay and the results highly correlated with the ELISA data (R = 0.81–0.98; Supplementary Table S1). Furthermore, in general, a clear positive trend was observed between the _BvLz__m_ copy

number, the combinatorial promoter-_BvLz__m_ cassettes transformed and the BvLzm levels (Table 2; Fig. 2; Supplementary Fig. S2). For instance, quadruple and triple promoter:_BvLz__m_ lines

displayed a higher _BvLz__m_ copy number and yield than double and single promoter:_BvLz__m_ lines, as expected from co-transformation (Table 2; Fig. 2; Supplementary Fig. S2). Similarly,

the double promoter pUD:_BvLz__m_ lines had a higher _BvLz__m_ copy number and accumulation than single promoter pU:_BvLz__m_ lines (Table 2; Fig. 2; Supplementary Fig. S2). The BvLzm yield

from single promoter pUbi:_BvLz__m_ (pU:_BvLz__m_) lines varied from low (0.08–0.1 mg/kg; 6.7% of plants) to moderate (0.12–0.18 mg/kg; 40.0% of plants) and high (0.2–0.4 mg/kg; 53.3% of

plants) (Tables 1, 2). The BvLzm yield range was 0.08–0.4 mg/kg (0.01–0.06% of TSP), averaging 0.3 mg/kg (0.04% of TSP) ± 0.02 for the high expressers (Table 1). Other single

promoter:_BvLz__m_ lines harboring pSHDIR16, pSCBV21, pSHPRP or pSHEF1α showed similar trends, with a highest BvLzm yield of 0.56 mg/kg (0.08% of TSP) (Supplementary Table S2). Stacked

double promoter pUbi-SHDIR16:_BvLz__m_ (pUD:_BvLz__m_) lines displayed 1.8–6.3 fold higher BvLzm yield than single promoter pU:_BvLz__m_ lines, with a range of 0.5–0.7 mg/kg (0.07–0.1% of

TSP) (Table 1). The BvLzm yield was further enhanced to 2.0–8.6 fold in the stacked triple promoter:_BvLz__m_ lines, with levels ranging from 1.0 to 6.0 mg/kg (0.1–0.8% of TSP) (Table 1).

The majority (66.7%) of the stacked triple promoter pUbi-SHPRP-SHEF1α:_BvLz__m_ (pUPE:_BvLz__m_) lines had a BvLzm yield of 1.0–2.0 mg/kg (0.1–0.3% of TSP) with 20.0% at 2.2–3.2 mg/kg

(0.33–0.45% of TSP) and 13.3% at 3.5–4.7 mg/kg (0.5–0.7% of TSP) (Table 1). Replacing the constitutive SHPRP promoter with the culm-regulated SHDIR16 promoter in the stacked triple promoter

pUbi-SHDIR16-SHEF1α:_BvLz__m_ (pUDE:_BvLz__m_) lines boosted the BvLzm yield to 6.0 mg/kg (0.8% of TSP). Most of pUDE:_BvLz__m_ lines (62.0%) had a BvLzm yield of 2.2–3.2 mg/kg (0.33–0.45%of

TSP), with 27.0% at 1.5–2.0 mg/kg (0.2–0.3% of TSP), 4.5% at 3.5–4.7 mg/kg (0.5–0.7% of TSP) and 6.5% at 5.0–6.0 mg/kg (0.7–0.8% of TSP) (Table 1). Next, we checked if stacking another

promoter to produce quadruple promoter:_BvLz__m_ lines would be helpful. The BvLzm yield increased modestly in the stacked quadruple promoter pUbi-SHPRP-SCBV21-SHEF1α:_BvLz__m_

(pUPBE:_BvLz__m_) lines by 1.7–2.4 fold, compared to the stacked triple promoter:_BvLz__m_ lines. The highest enhancement was achieved when using a double terminator cassette, i.e. 10.0 

mg/kg (1.4% of TSP) in pUPBE:_BvLz__m_:35STNOST lines (Table 1), and the 3′UTR of SrMV with the single 35S terminator, i.e. 6.3 mg/kg (0.9% of TSP) in pUPBE:_BvLz__m_:3′UTR35ST lines (Table

1). In fact, 24.1% of pUPBE:_BvLz__m_:35STNOST plants had a BvLzm yield of 6.0–10 mg/kg (0.8–1.4% of TSP), and 44.5% of pUPBE:_BvLz__m_:3′UTR35ST plants showed a BvLzm yield of 6.0–6.3 mg/kg

(0.8–0.9% of TSP) (Table 1). Lastly, we evaluated if event stacking can enhance the yields of the stacked quadruple promoter lines. Event stacking, also referred to as super transformation,

is a good alternative to hybridization/crossing, which is time-consuming and not a viable option in vegetatively-propagated crops like sugarcane. Stacked five promoter

pUbi-SHDIR16-SHEF1α-SHPRP-SCBV21:_BvLz__m_ (pUDEPB:_BvLz__m_) lines were generated through event stacking, by re-transforming bialaphos-resistant triple promoter pUDE:_BvLz__m_ lines with

two promoter:_BvLz__m_ expression vectors, pP:_BvLz__m_ and pB:_BvLz__m_ (Table 1) using the _neomycin phosphotransferase II_ as a selectable marker. The resulting pUDEPB:_BvLz__m_ lines

showed increased BvLzm accumulation, i.e. up to 82.5 mg/kg culm mass (11.5% of TSP) (Table 1). The majority (33.3%) of these lines exhibited BvLzm levels of 26.2–32.3 mg/kg (3.6–4.5% of

TSP), while 18.3% accumulated the highest BvLzm levels, i.e. 59.9–82.5 mg/kg (8.3–11.5% of TSP). The remaining 24.2% and 12.1% of the lines showed BvLzm levels of 15.9–21.1 mg/kg (2.2–2.9%

of TSP) and 11.0–12.4 mg/kg (1.5–1.7% of TSP), respectively (Table 1). Notably, BvLzm accumulation was highly enhanced in the new stacked five promoter pUDEPB:_BvLz__m_ lines by

7.3–13.8-fold, compared to the receiving stacked triple promoter pUDE:_BvLz__m_ lines. Together, these experiments demonstrate that high levels of recombinant BvLzm (up to 11.5% of TSP or

82.5 mg/kg) can be successfully produced in sugarcane culms using the combinatorial promoter and event stacking strategies. Previous studies utilized multiple plant species, tissue types,

and expression systems for recombinant protein production40,41. Majority of them used transient _Agrobacterium_- and viral vector-based approaches in _Nicotiana benthamiana_ or _N.

tabacum_42,43,44,45,46. While the transient systems are viable approaches, they are technically feasible only in few plant species that are amenable for infiltration and/or are hosts for the

viruses used as viral vectors. In this context, transgenic plant systems are more suited for wider adoption since broad range of plant species can be transformed using latest biotechnology

tools. When comparing our results of protein expression in sugarcane culms with other transgenic plant expression systems, caution was exercised particularly when comparing recovered protein

yields per starting tissue weight (e.g., mg/kg). This is because not all plant tissues have similar compositions, nor the protein extractions are equally efficient among tissue types, owing

to biological and biochemical differences41. For instance, sugarcane culms primarily constitute juice (sugars) and lignocellulosic fiber (bagasse). An equal amount of _N. benthamiana_

leaves on a fresh weight basis will have less fiber, and proteins may be easier to extract from leaf tissues. We also note that biochemical properties of target proteins such as size,

solubility, amino-acid composition, structural features, and protein stability may also ultimately influence the final yield. With these caveats in mind, we compared our results with other

reported studies of transgenic plant systems using the % TSP unit of recovered proteins. Several studies have reported recombinant protein yields of ~ 0.002 to 0.05% of TSP in transgenic

carrots47,48, ~ 0.23–2.5% of TSP in transgenic tobacco and potato49, ~ 8% TSP in transgenic tomato50, and ~ 11.9% in transgenic rice51. These comparisons suggest that higher protein yields

can be achieved using the sugarcane transgenic system (up to 11.5% of TSP), which are comparable to other transgenic systems, if not greater. In addition to the use of constitutive or

tissue-specific promoters, inducible promoters can be used for expressing recombinant proteins in plants52,53,54. Several inducible promoters can be used for generating transgenic plants

such as dexamethasone-, ethylene-, heat shock- and estradiol-inducible promoters52. Indeed, we have previously shown that the sugarcane DIRIGENT (SHDIR16) promoter is responsive to plant

hormones such as salicylic acid or jasmonic acid35. This is promising and suggests that inducible promoters such as SHDIR16, and other well-characterized plant inducible-promoters52 can be

further used in lieu or in combination with the constitutive/tissue-specific promoters that we have described, in order to robustly control and/or fine-tune the recombinant protein

expression. INCREASED PROTEIN LEVELS WERE ASSOCIATED WITH THE NUMBER OF COMBINATORIAL STACKED PROMOTERS AND NOT WITH THE COPY NUMBER ALONE Our results show that using multiple different

promoters to drive expression of recombinant _BvLz__m_ on distinct vectors enhanced recombinant protein accumulation. It is possible that the enhanced levels may have occurred due to higher

number of inserted _BvLz__m_ copies alone or it could be due to a combination of promoter-driven synergistic transcriptional activity. To test these scenarios, we performed a comparison of

the BvLzm transcript and yield among the various promoter stacked lines that had similar number of insertions. This analysis showed that there is a positive correlation in BvLzm transcript

and yield with combinatorial promoter:_BvLz__m_ stacks, irrespective of the number _BvLz__m_ inserts (Fig. 2; Supplementary Fig. S2). For instance, for single promoter:_BvLz__m_ line 13,

double promoter:_BvLz__m_ line 42, triple promoter:_BvLz__m_ line 20 and quadruple promoter:_BvLz__m_ line 10, with all of them having about 4–5 _BvLz__m_ inserts, there was a clear

enhancement in the BvLzm yield (Fig. 2a,c; Supplementary Fig. S2). Conversely, a comparison of single promoter:_BvLz__m_ transgenic lines with one or multiple inserts showed that there was

no corresponding increase in BvLz yield with the copy number. For instance, line 19 with one insert (Fig. 2a,c; Supplementary Fig. S2) had a BvLzm yield of 0.2 mg/kg, while line 13 with 4

_BvLz__m_ inserts had a BvLzm yield of 0.15 mg/kg (Fig. 2a, c; Supplementary Fig. S2). Together, these results suggest that the increase in BvLzm yield is primarily attributed to the number

of combinatorial stacked multiple promoters and not just with the _BvLz__m_ copy number alone. COMBINATORIAL PROMOTER STACKING MAY ALLEVIATE TRANSCRIPTIONAL OCCLUSION AND/OR RECOMBINANT GENE

SILENCING Multiple identical copies of recombinant genes or promoter transcription units (PTUs) delivered through a single construct could trigger transgene silencing55,56,57 or result in

promoter occlusion or transcriptional interference, a phenomenon observed in eukaryotic systems, including plants58,59,60,61,62. For instance, a strong PTU can sequester most of the

transcription factors in its immediate vicinity, limiting transcription from other promoters present in _cis_ on the same vector63. Alternatively, homology-dependent DNA methylation within

the promoter or in the coding region sequences could result in transgene silencing. For instance, in maize, transgenic lines with four copies of a cellulase gene, under control of tandemly

arranged PTUs on the same vector, resulted in lowered expression than those lines with fewer copies64. Our results here showed a positive correlation between the number of combinatorial

promoter stacks of recombinant _BvLz__m_ and increase in _BvLz__m_ levels, with no apparent transgene silencing. It is likely that using different promoter sequences in separate vectors may

overcome the transgene silencing or transcriptional interference. We suggest that each expression vector in the described stacked multiple promoter:_BvLz__m_ system (Fig. 1) does not

negatively affect the others, as shown by a positive correlation between the combinatorial promoter:_BvLz__m_ copy number (Table 2) and enhanced steady-state _BvLz__m_ transcript

accumulation (Fig. 2b; Supplementary Fig. S3) and BvLzm activity (Table 1; Fig. 2c). ELEVATED RECOMBINANT BVLZM ACCUMULATION POSITIVELY ENHANCES TRANSGENIC PLANT GROWTH Analysis of the

deleterious effects of recombinant protein accumulation on plant physiology and growth is crucial in order to assess the economic feasibility of using transgenic plants as biofactories, and

this is largely dependent on the target protein function65. In our scenario with BvLzm, we found no deleterious effects of enhanced _BvLz__m_ expression on sugarcane growth. On the contrary,

several growth characteristics of _BvLz__m_ expressing lines were better than those of non-transformed plants, such as enhanced leaf length, culm height, tiller number, culm biomass and

Brix (total soluble solids) (Table 3). These differences were statistically significant (_p_ < 0.001 and _p_ < 0.0001) in the triple and quadruple promoter:_BvLz__m_ expressing lines

(Table 3). For instance, mean culm fresh biomass per plant of the quadruple promoter:_BvLz__m_ expressing lines was nearly 2.5 times greater than that of non-transformed plants. The mean

soluble solids content in juice from triple and quadruple promoter:_BvLz__m_ expressing lines was approximately 20% higher than that of non-transformed plants. Similar trends were also

observed for leaf length, culm height and tiller density. The enhanced agronomic performance of the transgenic lines suggested that BvLzm, which is a well-known antimicrobial protein27,

could have a growth-promoting or perhaps protective role against pathogens present in the natural growth environment. RECOMBINANT PROTEIN ACCUMULATION IN CULMS INCREASES WITH PLANT AGE To

monitor the temporal stability of BvLzm accumulation in sugarcane culms in a growing season, we analyzed BvLzm levels for 11-months with a selection of several representative single promoter

pU:_BvLz_m lines. The BvLzm yields (mg of BvLzm/kg of harvested culm) in these lines after 7-, 9- and 11-month-harvest are shown in Fig. 4 (data for four representative lines) and Table 3

(data for six representative lines at the 11 month-harvest). There was a significant (_p_ < 0.05) increase in BvLzm yield over time for all the lines tested. BvLzm accumulation was

highest at the 11-month harvest, with lines 67, 108 and 114 showing the most significant (_p_ < 0.05) increase (Fig. 4). This accumulation pattern coincides with timing of culm ripening,

which is characterized by increased sucrose translocation and accumulation in culms. The age-related sucrose accumulation also was associated with the reduction in vegetative development

(leaf initiation and expansion) and commences at the mature basal internodes, progressing towards the culm apex, until the entire culm reaches a stable sugar level as it approaches

physiological maturity66. The age-related pattern of BvLzm accumulation may also be regulated by similar factors whereby photoassimilates and other substrates for BvLzm are diverted from

vegetative growth towards metabolite synthesis and accumulation during sugarcane maturation. Regardless of the mechanisms regulating the temporal accumulation of BvLzm, our results

demonstrate that the recombinant protein levels can be maintained, if not enhanced, during the development phases of sugarcane in a growing season. Similar results were observed for BvLzm

accumulation in representative triple promoter:_BvLz_m lines, which showed sustained and stable BvLzm levels over a full growing year, as well as in successive vegetative propagations

(Supplementary Table S3). Similar accumulation of recombinant proteins (human therapeutic interleukin-10) with plant maturity was observed in tobacco67. HIGH LEVEL RECOMBINANT PROTEIN

ACCUMULATION REQUIRES ADEQUATE MINERAL NUTRITION TO SUSTAIN THE PROTEIN AND BIOMASS ACCUMULATION Adequate water and nutrients supply are important for crop productivity as well as quality

considerations, such as protein content and other sensory traits68. Because we observed enhanced growth traits such as biomass in the _BvLz__m_ expressing lines, specifically in the triple

promoter:_BvLz__m_ lines (Table 3), we next investigated the optimal fertilization regime needed to sustain the additional growth and high levels of BvLzm production. Four representative

triple promoter:_BvLz__m_ lines (2-month old) were subjected to two mineral nutrient supply regimes namely, low fertility (LF or 2.4 mg N per plant, twice a week) and a high fertility (HF or

8 mg N per plant), using a balanced commercial fertilizer (Peters Professional 20–20–20; see “Materials and methods” section). BvLzm yield and growth traits were measured at 2-, 6-, and

8-months following fertilization. Supplemental fertilization increased culm biomass and BvLzm yield in the triple promoter:_BvLz__m_ lines over time. The most significant increases (_p_ <

 0.05) between LF and HF were noted at 2 months for all lines (Fig. 5). For instance, pUPE:_BvLz__m_ line 32C (CP72-1210 variety) and pUDE:_BvLz_m lines 19, 44 and 54 (TCP98-4454 variety)

showed 4.6-, 2.5-, 3.0- and 2.0-fold increases in culm biomass and 1.7-, 1.3-, 1.1 and 1.0-fold enhancements in BvLzm yield, respectively. Leaf macronutrient contents of the triple

promoter:_BvLz__m_ plants were also monitored following growth under the two fertilization regimes. Plants grown under high nutrient supply rates had significantly (_p_ < 0.0001) higher

leaf mineral nutrient contents compared to those grown under low nutrient supply rates (Table 4). Leaves of HF plants had higher levels of N, phosphorus (P), potassium (K) and magnesium

(Mg), compared to leaves of LF plants (Table 4). In general, leaf nutrient content of the _BvLz__m_ expressing lines was improved by supplemental fertilization, resulting in a 1.5- to

2.2-fold increase in culm biomass and a subsequent 1.2- to 2.2-fold enhancement in BvLzm yield at 8 month-growth stage (Fig. 5). Taken together, the accumulation of _BvLz__m_ in response to

fertilization and the ontogenic _BvLz__m_ accumulation pattern underscore the need for adequate input availability to sustain not only biomass production but also the yield of high-value

proteins in crops such as sugarcane. CONCLUSIONS The genetic/biotechnology tools and resources developed in this study not only expands the utility of sugarcane for large-scale production of

recombinant proteins but can be utilized with other monocots and bioenergy feedstocks. Our approach comprises stacking multiple promoters to co-express codon-optimized recombinant genes

from different expression vectors using combinatorial transformation methods. This resulted in high recombinant protein yield (up to 11.5% of TSP or 82.5 mg/kg) in transgenic culms,

rendering it an attractive biopharming tool for potential commercial uses69. We also showed that recombinant BvLzm levels can be maintained stably throughout the growing season and had no

negative consequences on sugarcane agronomic performance. Overall, our study provides new knowledge, tools and resources to expand the utility of sugarcane beyond a food crop and bioenergy

feedstock to using it as a biofactory for expressing high-value proteins25. MATERIALS AND METHODS EXPRESSION VECTORS BASIC VECTORS A series of expression vectors were constructed, using a

custom synthesized bovine lysozyme (_BvLz_) gene codon-optimized for expression in maize (_BvLz__m_) (444.0 base pairs [bp])39 (GenScript, Piscataway, NJ). The _BvLz__m_ gene was subcloned

into pUC57 at _Bam_HI and cloned at the same site into pZero2 (Invitrogen, ThermoFisher Scientific, Waltham, MA), to which the 35ST34,38,39 (197.0 bp) was added at the _Pst_I site, resulting

in the _BvLz__m_-35ST/pZero2 plasmid. Three basic _BvLz_ expression vectors were generated with the constitutive promoters pUbi33, pSHPRP36 or pSHEF1α36. The first vector,

pUbi-_BvLz__m_-35ST/pZero2 was produced by cloning the pUbi fragment (1,977 bp), released from pAHC20 (pUbi:_BAR_/pUC8)70 (pUbi minus heat shock element; a 28.0 bp deletion at the 5′ end of

pUbi) with _Bam_HI/_Hind_III and filled in, into the filled-in _BvLz__m_-35ST/pZero2. For the other two vectors, the _Sma_I-treated pSHPRP (3,016 bp) and pSHEF1α (1,959 bp) fragments from

pSK+36 were fused to the _Sna_BI/_Bbs_I-treated/filled-in _BvLz__m_-35ST fragment from pUbi-_BvLz__m_-35ST/pZero2 to yield pSHPRP-_BvLz__m_-35ST/pSK+ and pSHEF1α-_BvLz__m_-35ST/pSK+,

respectively. Two basic _BvLz_ expression vectors were generated with the culm-regulated promoters pSHDIR1635 or pSCBV2134. The pSHDIR16-_BvLz__m_-35ST/pSK+ vector was assembled by fusing

_BvLz__m_-35ST, excised from _Bam_HI/_Eco_RI-treated _BvLz__m_-35ST/pZero2, to the pSHDIR16 fragment35 (2,680 bp) at the same sites in pSK+. The pSCBV21-_BvLz__m_-35ST/pGEMT-T Easy vector

was produced by cloning _BvLz__m_-35ST, excised from _Bam_HI/_Eco_RI-treated _BvLz__m_-35ST/pZero2, into the _Nco_I-treated/filled-in pSCVB21 (1,816 bp)/pGEM-T Easy34. DOUBLE TERMINATOR

VECTORS _BvLz_ constructs with a double terminator were generated by fusing the NOST (253 bp)39 to the 35ST of basic _BvLz_ constructs. The pUbi-_BvLz__m_-35STNOST/pZero2 vector was

constructed by releasing the NOST from pBI221 (Accession Number AF502128) (Clontech Laboratories, Inc., Mountain View, CA) with _Eco_RI/_Sst_I, filled in and cloned into the

_Xho_I-treated/filled-in pUbi-_BvLz__m_-35ST/pZero2. To make pSHPRP-_BvLz__m_-35STNOST/pSK+ and pSHEF1α-_BvLz__m_-35STNOST/pSK, the _Sna_BI/_Bbs_I-treated/filled-in _BvLz__m_-35STNOST

fragment from pUbi-_BvLz__m_-35STNOST/pZero2 was fused to the _Sma_I-treated pSHPRP/pSK+ and pSHEF1α/pSK+ vectors, respectively. To generate the pSCBV21-_BvLz__m_-35STNOST/pGEM-T Easy

vector, the _Sna_BI/_Bbs_I-treated/filled-in _BvLz__m_-35STNOST fragment from pUbi-_BvLz__m_-35STNOST/pZero2 was cloned into _Nco_I-treated/filled-in pSCVB21/pGEM-T Easy. VECTORS WITH VIRAL

UNTRANSLATED REGIONS The 3′UTR of SrMV strain H (GenBank Accession Number U57358) (235.0 bp) was custom synthesized as a fusion to _BvLz__m_ in pJI (_BvLz__m_-SrMV 3′UTR/pJI) (ATUM, DNA2.0,

Newark, CA). The pUbi-_BvLz__m_-SrMV 3′UTR-35ST/pZero2 vector was assembled by cloning the filled-in SrMV 3′UTR, released from _Eco_RI/_Bgl_II-treated _BvLz__m_-3′SrMV/pJI, into

pUbi-_BvLz__m_-35ST/pZero2 at the _Sma_I site. The pSHPRP-_BvLz__m_-SrMV 3′UTR-35ST/pSK+ and pSHEF1α-_BvLz__m_-SrMV 3′UTR-35ST/pSK+ vectors were generated by fusing the

_Sna_BI/_Bbs_I-treated/filled-in _BvLz__m_-SrMV 3′UTR-35ST fragment from the pUbi-_BvLz__m_-SrMV 3′UTR-35ST/pZero2 to pSHPRP/pSK+ and pSHEF1α/pSK+ at the _Sma_I site, respectively. For

construction of pSHDIR16_-BvLz__m_-SrMV 3′UTR-35ST/pSK+ vector, SrMV 3′UTR was released from _BvLz__m_-SrMV 3′UTR/pJI by _Eco_RV treatment and cloned into pSHDIR16-_BvLz__m_-35ST/pSK+ at the

_Eco_RV site. All DNA cloning steps were carried out as described by Sambrook71. Filling in of endonuclease-treated DNA fragments and dephosphorylation of vectors were done using T4 DNA

polymerase (NEB BioLabs, Ipswich, MA) and antarctic phosphatase (NEB BioLabs), respectively. SUGARCANE TRANSFORMATION Tops of field-grown sugarcane (_Saccharum_ spp. hybrids) commercial

varieties CP72-1210, CP84-1198, TCP87-3388 and TCP98-4454 were collected during the growing season, and leaf roll discs were prepared for stable transformations as previously described72.

Briefly, leaf blades and sheaths were removed down to the top visible dewlap leaf, and the upper 20–30 cm portion of shoot (leaf roll culm) was surface sterilized in 70.0% (v/v) ethanol for

20 min. Immature leaf rolls close to the apical meristem were sliced transversely into 1.0 mm thick sections and cultured on MS3 medium (MS medium with 3.0 mg/l of 2,4-dichlorophenoxyacetic

acid [2,4-D]) for 30–35 days (for embryogenic calli) or MS0.6 medium (MS with 0.6 mg/l of 2,4-D) for 7–10 days (for embryogenic leaf roll discs). Embryogenic calli and leaf roll discs were

preconditioned on MS3- and MS0.6-osmoticum (MS3 or MS0.6 with 0.2 M d-mannitol and 0.2 M d-sorbitol), respectively, for 4 h before and after DNA particle bombardment. DNA bombardment was

performed according to Beyene and colleagues38. Briefly, tungsten particles (1.1 µm; Bio-Rad Laboratories, Inc.) (1.0 mg) were coated separately with plasmid DNA (1.0 µg) of different

constructs at equimolar ratios together with pUbi:_BAR_/pUC8 selectable marker plasmid using calcium chloride (NaCl) (1.0 M) and spermidine (14.0 mM). The DNA particle suspension (containing

the selectable marker plasmid with one or more _BvLz__m_ plasmids) (4.0 μl; 0.5 µg DNA per bombardment) was placed at the center of a syringe filter and delivered into tissue with a

particle inflow gun using a 26.0-inch Hg vacuum and a 7.0-cm target distance. Bombarded embryogenic calli and leaf roll discs were maintained on MS3 and MS0.6, respectively, for 10 days in

the dark at 28 °C for recovery. They were later incubated in the dark at 28 °C on selection medium (MS3 or MS0.6 with bialaphos at 3.0 mg/l) for a total of 2 weeks. Shoot regeneration and

root initiation were performed under bialaphos selection as previously described72. Rooted plantlets were transferred to potting soil (Sunshine Mix #1; SunGro Horticulture Distribution,

Inc., Agawan, MA) in pots and maintained in the greenhouse. TRANSGENIC PLANT SCREENING INTEGRATION AND SIZE DETERMINATION OF _BVLZ__M_ EXPRESSION CASSETTES Integration and size of each

_BvLz__m_ expression cassette in the single and multiple stacked promoter:_BvLz__m_ sugarcane lines were determined by Southern blot and PCR analyses, respectively, using genomic DNA

isolated according to Tai and Tanksley73 from liquid N-ground tissues (3.0 g) collected from young leaves of 3–4 month-old plants. Controls included vector-transformed lines and

non-transformed plants (tissue culture-derived). For Southern blot analysis, genomic DNA (10.0 μg per lane) was treated with _Hind_III endonuclease, electrophoresed on 0.8% (w/v) agarose

gels and transferred to nylon membranes (Amersham Hybond-XL, GE Healthcare Bio-Sciences Corp., Piscataway, NJ) in 0.4 M sodium hydroxide74. Pre-hybridization, hybridization, washing and

detection of DNA gel blots were performed using Church’s buffer75. The probe, corresponding to the _BvLz__m_ coding sequence was amplified by PCR from pUbi-_BvLz__m_-35ST/pZero2 using the

primer set BvLz-1F (5′-ATGGCGGCCCTGGTGATCCTGGGCT-3′) and BvLz-481R (5′-TCACAGGGTGCAGCCTTCCACG-3′) and labeled with [α-32P] dCTP using the Random DNA Labeling kit (Invitrogen, ThermoFisher

Scientific). PCR was performed on a C1000 Touch thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA) in a total reaction volume of 25.0 µl using 200.0 ng of DNA and Platinum _Taq_ DNA

polymerase (Invitrogen, ThermoFisher Scientific) according to the manufacturer’s instructions with the following conditions: 94 °C for 4 min, 35 cycles each at 94 °C for 30 s, 49.7–54.4 °C

for 30 s, and 72 °C for 6 min. Primers encompassing the entire promoter:_BvLz__m_-terminator cassette (Supplementary Table S4) were designed with Primer 3.0. All PCR amplicons were separated

by electrophoresis on 0.7% agarose (w/v) gels stained with ethidium bromide. A “no DNA template” was included as a negative control for PCR. DETERMINATION OF _BVLZ__M_ COPY NUMBER _BvLz__m_

copy number in single and multiple stacked promoter:_BvLz__m_ sugarcane lines was estimated by qPCR. qPCR was performed on a CFX384 Real-time PCR Detection System (Bio-Rad Laboratories,

Inc.) using i_Taq_ Universal SYBR Green Supermix (Bio-Rad Laboratories, Inc.), 0.4 µM of each target specific primer and 1.0 ng of genomic DNA from representative transgenic _BvLz__m_ lines,

according to the manufacturer’s instructions. Primers specific to the promoter-_BvLz__m_ gene junction area (Supplementary Table S4) were designed with Primer 3.0

(https://bioinfo.ut.ee/primer3-0.4.0/primer3/). qPCR conditions were as follows: 95.0 °C for 3 min, 39 two-step cycles each at 96.0 °C for 5 s and 57 °C for 30 s, and a final melting curve

of 60.0 °C to 95.0 °C for 6 min. The sugarcane anthranilate phosphoribosyltransferase and prolyl 4-hydroxylase genes were used as a reference for single copy genes76. qPCR was performed

twice in triplicate with two biological replications. PCR efficiency was calculated with LinReg77. Results were analyzed and recorded as CT (threshold cycle) values. Copy number of the

_BvLz__m_ gene was estimated by qPCR according to Casu et al.76 using the formula GCI = EffRefCT/EffCT, where: GCI = gene copy number index, EffRefCT = PCR efficiency using the reference

gene primers to the power of the reference gene CT value for each sample, and EffCT = PCR efficiency using the test gene primers to the power of the test gene CT value generated for each

sample. EXPRESSION ANALYSIS OF _BVLZ__M_ Total RNA was isolated by grinding 1.0 g of young leaves collected from 3–4 month-old plants in liquid N39,78. For northern blot analysis, RNA (15.0 

μg per lane) was fractionated on 1.6% formaldehyde agarose denaturing gels in HEPES buffer and blotted onto nylon membranes (Amersham Hybond-XL) in 10x SSC75. Pre-hybridization, _BvLz__m_

probe labeling, hybridization, washing and detection of RNA gel blots were performed as described for Southern blot analysis. PLANT GROWTH AND TREATMENT CONDITIONS For growth cycle

investigations, single-node culm cuttings of 15 single promoter pU:_BvLz__m_ transgenic lines and non-transformed plants were pre-germinated in seedling flats (Supplementary Fig. S1) for 2.5

 weeks and transplanted into 37.0-l pots (four pots per line) in commercial growth medium (Sunshine Mix #1). Plants were maintained in a temperature-regulated greenhouse with average

day/night temperatures of 32/22 °C and relative humidity of 60–100%. Plants were initially fertilized once per week with a commercial high-phosphorus soluble fertilizer (Peters

8%N-19.8%P-12.5%K; The Scotts Company, Marysville, OH) for 5 weeks and then with a balanced/complete soluble fertilizer (Peters Professional 20–20–20; The Scotts Company) containing N 200.0 

g/kg, P 80.0 g/kg, K 166.0 g/kg, Mg 1.0 g/kg, iron 0.5 g/kg, manganese 0.3 g/kg, boron 0.1 g/kg, copper 0.13 g/kg, molybdenum 0.05 g/kg, and zinc 0.25 g/kg. To assess the impacts of mineral

nutrient supply on growth and BvLzm accumulation, plants from four representative triple promoter pUDE:_BvLz__m_ lines and one representative triple promoter pUPE:_BvLz__m_ line were

pre-germinated and transplanted into 15.0-l plastic pots containing the same growth medium as described above. All pots were initially fertilized with a high-phosphorus fertilizer (Peters

8%N–19.8%P–12.5%K; Scotts, Marysville, OH; equivalent to 10.0 kg N/ha). After 2 months, pots were randomly assigned into two fertilization treatment groups, namely, high fertility (HF) and

low fertility (LF), with four pots per line selected for each group. Non-transformed plants (tissue culture-derived) were included as negative controls. Fertilization treatments were

achieved with a complete fertilizer (Peters Professional 20–20–20) containing macro- and micro-nutrients as described above. Plants in the LF group received an additional equivalent of 20.0 

kg N/ha whereas HF plants received 50.0 kg N/ha from supplemental fertilization using Peters Professional 20–20–20 (described above). Fertilizer treatments were applied in split doses (twice

per week). Transgenic culms were harvested at 2, 6 and 8 months following fertilization, processed, and their BvLz yield was determined by ELISA at the BioSeparation Facility of Texas

A&M University’s Biological and Agricultural Engineering Department (College Station, Texas). PLANT PHYSIOLOGICAL ANALYSIS For inorganic mineral analysis, leaf tissue samples were

collected, dried (70 °C for 48 h), ground to pass a 40-μm screen and analyzed for inorganic minerals. Total Kjeldahl N (ammonia and organic N) was determined in digested samples using the

EasyChem Plus Analyzer and protocols (Systea Scientific, Chicago, IL), whereas other macronutrients such as P, K and Mg were analyzed using the Optima 7300 DV Inductively Coupled

Plasma-Optical Emission Spectrometer (PerkinElmer, Shelton, CT) after partial digestion (hydrolysis) on a HotBlock Digestion System (Environmental Express, Inc., Charleston, SC). TOTAL

PROTEIN EXTRACTION Large-scale extraction and size fractionation of total soluble proteins (TSPs) from culms (300.0 lbs) of _BvLz__m_ transgenic sugarcane were performed at our Pilot Plant

Facility mainly as described previously79. Bench-scale extraction and purification of BvLzm from extracts of transgenic sugarcane culms (100.0 g), using a single-step hydrophobic interaction

chromatography, were performed at our BioSeparation Facility (College Station, Texas) as previously described30. For small-scale extraction of TSP from _BvLz__m_ transgenic sugarcane leaf

tissue (200.0 mg) was homogenized in 600.0 µl of sodium acetate buffer (50 mM NaOAc, pH 4.4, 0.1 M NaCl) in 2.0 ml tubes for 30 s at 5,000 rpm with the Precellys 24 homogenizer (MO BIO

Laboratories, Carlsbad, CA) using ceramic spherical beads (0.64 cm-diameter). TSP supernatants were collected by centrifugation at 13,000_g_ for 25 min at 4 °C. DETERMINATION OF BVLZM

ACCUMULATION BY ENZYME ACTIVITY AND ENZYME-LINKED IMMUNOSORBENT ASSAYS To determine the levels of recombinant BvLzm, enzyme activity and enzyme-linked immunosorbent assays (ELISA) were

performed on TSP from culm extract juice. Juice was extracted from 1.0 kg of culms of greenhouse grown _BvLz__m_ transgenic plants at 7, 9 and 11 months for the growth cycle experiment and

at 2, 6 and 8 months for the fertilization experiment. For enzyme activity determination, culm extract juice was tested for its ability to lyse _Micrococcus lysodeikticus_ cells using the

standard protocol from Sigma-Aldrich (St. Louis, MO). Rabbit anti-BvLz antibody used in the ELISA was synthesized by Bethyl Laboratories, Inc. (Montgomery, TX) using tobacco-derived BvLz31

and further purified through an SP-Sepharose column (GE Healthcare, Piscataway, NJ). ELISA of culm extract juice was performed as previously described30. Briefly, a sandwich ELISA consisting

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_Biotechnology_7, 160 (1989). CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We would like to dedicate this manuscript to late Professor T. Erik Mirkov (1959–2018), a pioneer in

sugarcane biotechnology. We gratefully acknowledge Xavier Gonzales for the assembly of three genetic constructs, Renesh Bedre for the statistical analysis of data and Sonia Irigoyen (Texas

A&M AgriLife Research) for critical review of the manuscript. We are grateful to Denise Rossi, Hyun Park Kang, Ninfa Ramos, Soledad Al-Varez, Gerleene Acuna, Adan Solis and Abigail Cruz

for excellent technical assistance. This research was supported by funds from BioCane, Inc., a subsidiary of Grower Research Group LLC (Soledad, CA) and Texas A&M AgriLife Research

Grants (124738-96210; 124190-96210) to K.K.M. AUTHOR INFORMATION Author notes * Carol Vargas-Bautista Present address: College of Medicine, Texas A&M University, 8447 Riverside Parkway,

Bryan, TX, 77807, USA AUTHORS AND AFFILIATIONS * Texas A&M AgriLife Research and Extension Center, 2415 East US Highway 83, Weslaco, TX, 78596, USA Mona B. Damaj, John L. Jifon, Carol

Vargas-Bautista, Joe Molina & Kranthi K. Mandadi * Department of Horticultural Sciences, Texas A&M University, College Station, TX, 77843-2133, USA John L. Jifon * National Center

for Therapeutics Manufacturing, Texas A&M University, 100 Discovery Drive, College Station, TX, 77843-4482, USA Susan L. Woodard * BioSeparation Laboratory, Biological and Agricultural

Engineering Department, College Station, TX, 77843-2117, USA Georgia O. F. Barros, Steven G. White & Zivko L. Nikolov * Innovus Pharmaceuticals, Inc., 8845 Rehco Road, San Diego, CA,

92121, USA Bassam B. Damaj * Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX, 77843-2132, USA Kranthi K. Mandadi Authors * Mona B. Damaj View

author publications You can also search for this author inPubMed Google Scholar * John L. Jifon View author publications You can also search for this author inPubMed Google Scholar * Susan

L. Woodard View author publications You can also search for this author inPubMed Google Scholar * Carol Vargas-Bautista View author publications You can also search for this author inPubMed 

Google Scholar * Georgia O. F. Barros View author publications You can also search for this author inPubMed Google Scholar * Joe Molina View author publications You can also search for this

author inPubMed Google Scholar * Steven G. White View author publications You can also search for this author inPubMed Google Scholar * Bassam B. Damaj View author publications You can also

search for this author inPubMed Google Scholar * Zivko L. Nikolov View author publications You can also search for this author inPubMed Google Scholar * Kranthi K. Mandadi View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS M.B.D., J.L.J., S.L.W., C.V.-B., G.O.F.B., J.M., Z.L.N., and K.K.M. designed the experiments. B.B.D.

designed the combinatorial gene expression system and developed total protein enrichment protocols. M.B.D. made the genetic constructs and prepared the manuscript. M.B.D. and J.M. conducted

transformation experiments. M.B.D., J.M. and C.V.-B. conducted transgenic plant screening analyses. J.L.J. conducted growth cycle experiments. M.B.D. and J.M. conducted fertilization

experiments. S.L.W., G.O.F.B. and S.G.W. conducted protein extraction, ELISA and purification experiments. S.L.W., J.L.J., Z.L.N., and K.K.M. supervised the study and reviewed the

manuscript. CORRESPONDING AUTHORS Correspondence to Mona B. Damaj or Kranthi K. Mandadi. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL

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CITE THIS ARTICLE Damaj, M.B., Jifon, J.L., Woodard, S.L. _et al._ Unprecedented enhancement of recombinant protein production in sugarcane culms using a combinatorial promoter stacking

system. _Sci Rep_ 10, 13713 (2020). https://doi.org/10.1038/s41598-020-70530-z Download citation * Received: 06 December 2019 * Accepted: 21 July 2020 * Published: 13 August 2020 * DOI:

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