Bioprocess for hydrolysis of galacto-oligosaccharides in soy molasses and tofu whey by recombinant Pseudomonas chlororaphis

Journal Pre-proof Bioprocess for hydrolysis of galacto-oligosaccharides in soy molasses and tofu whey by recombinant Pseudomonas chlororaphis Daniel K.Y. Solaiman, Richard D. Ashby, Nicole V. Crocker PII:

S1878-8181(19)31696-2

DOI:

https://doi.org/10.1016/j.bcab.2020.101529

Reference:

BCAB 101529

To appear in:

Biocatalysis and Agricultural Biotechnology

Received Date: 1 November 2019 Accepted Date: 4 February 2020

Please cite this article as: Solaiman, D.K.Y., Ashby, R.D., Crocker, N.V., Bioprocess for hydrolysis of galacto-oligosaccharides in soy molasses and tofu whey by recombinant Pseudomonas chlororaphis, Biocatalysis and Agricultural Biotechnology (2020), doi: https://doi.org/10.1016/j.bcab.2020.101529. 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. © 2020 Published by Elsevier Ltd.

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Bioprocess for hydrolysis of galacto-oligosaccharides in soy molasses and tofu whey by recombinant Pseudomonas chlororaphis

4 Daniel K.Y. Solaiman* · Richard D. Ashby Nicole V. Crocker 6 Biobased and Other Animal Co-Products Research Unit, Eastern Regional Research Center, 8

Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA

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Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S.

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Department of Agriculture. USDA is an equal opportunity provider and employer.

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Correspondence to: D.K.Y. Solaiman, E-mail: [email protected].

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Abstract 24

The main objective of the study was to develop a bioprocess to hydrolyse galactooligosaccharides (GOs) using genetically engineered Pseudomonas chlororaphis which could

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produce biopolymers and biosurfactants. To this end, alpha-galactosidase genes were expressed in P. chlororaphis transformants either via a plasmid vector (i.e., strain [dAG]) or integrated into

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the chromosome (i.e., strain [chr::AG]). Pseudomonas secretion signals for protein were tested to facilitate extracellular secretion of the gene product. Furthermore, a two-stage condensed-cell-

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density fermentation process was adopted to enable hydrolysis of GOs by the P. chlororaphis recombinants. The results showed that the recombinant P. chlororaphis strains could hydrolyse

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GOs in culture media containing raffinose alone, stachyose alone, crude soy molasses, and tofu whey waste byproducts. Specifically, the most active recombinant P. chlororaphis [dAG] strain

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reduced the levels of raffinose (0.5% w/v) to <0.1% (w/v) and stachyose (0.21% w/v) to 0.15% (w/v) in E* medium after 7-d incubation. When used in E* medium containing soy molasses

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(SM), [dAG] strain reduced raffinose (0.1% w/v) and stachyose (0.56% w/v) to 0.03% w/v and 0.25% w/v, respectively, after a 7-d incubation. In E* medium containing tofu whey (TW),

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[dAG] reduced raffinose (0.11% w/v) and stachyose (0.27% w/v) to 0.05% w/v and 0.08% w/v, respectively, after an 8-d incubation. The other recombinant P. chlororaphis strain [chr::AG]

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was also capable of reducing GO levels in supplemented E* media but at a lower reactivity than the [dAG] strain (see detail in text).

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Keywords condensed-cell-density bioprocess; poly(hydroxyalkanoate); raffinose; rhamnolipid; stachyose; two-stage fermentation

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Introduction Development of low- or no-cost industrial and agricultural waste streams and byproducts for use

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as fermentation feedstocks is an attractive approach to help reduce the production costs of microbial products (Urnau et al., 2019). Among the potential low-cost feedstocks are the

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byproducts that contain carbohydrates which are useful as carbon and energy sources for microbial fermentation. Galacto-oligosaccharides (GOs) (i.e., raffinose, stachyose, and

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melibiose) are found in agricultural byproducts and processing waste streams, notably in soy molasses (SM), tofu whey (TW), and beet-sugar molasses byproduct (Kuo et al. 1988). With the

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ever-increasing consumer demands for plant-based food products and ingredients at the estimated annual growth rates of 5-10%, the availability of GO-containing waste streams is

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expected to increase accordingly, thus creating a bountiful supply of potential low-cost fermentative feedstocks. SM is generated as a heavy syrupy byproduct during the production of

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food-grade soy protein concentrate through an alcohol precipitation process. Half of the syrupy SM consists of soluble solids that include soy sugars (including the GOs), residual soy proteins,

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oils, and minerals. It is usually disposed of by spraying on animal feeds or farm soils (Chajuss 2004). TW is the liquid byproduct that is generated when chemically or enzymatically

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coagulated soymilk (McHugh 2016) is pressed and shaped into tofu curds. Although the major tofu consumer markets are in Asia, the popularity of plant-based protein diets in the Western

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world in recent years has resulted in high growth rates for the tofu market, leading to a high volume of TW being generated and thus presenting a low-cost feedstock alternative for

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fermentation processes. Aside from GO’s and other soy sugars (i.e., sucrose and glucose), the TW byproduct stream also contains residual soybean components as found in SM plus the added

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coagulants. Unlike SM, however, TW is usually treated and disposed into the municipal water-

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treatment system because tofu manufacturers are mostly located in or near major population 70

centers (Belén et al. 2012). Value-added utilization of TW such as in fermentation bioprocesses is thus an attractive proposition. Another food industrial byproduct that contains a sizeable

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amount of GO (i.e., raffinose at 1%; Šarić et al. 2016) is sugar-beet molasses. Sugar beet is a principal source of table sugar (i.e., sucrose) in the Western world with an estimated 2018-2019

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annual production of 4.9 million metric STRV (short tons, raw value) in the U.S. alone (McConnell and Olson 2018). Thus, in addition to SM and TW, sugar-beet molasses is also a

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large-volume byproduct stream that could potentially be used as a low-cost fermentation feedstock for bioprocesses.

78 GOs are hydrolyzed into their constituent monosaccharides before microorganisms can 80

metabolize and utilize them as a carbon and energy source. The two enzymes required for the hydrolysis of GOs into their monosaccharides are the α-galactosidase (α-D-galactoside

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galactohydrolase, EC 3.2.1.22) (α-Gal) and invertase (β-fructofuranosidase, EC 3.2.1.26) (Supplemental Figure S1). Some microorganisms possess the two enzymes and thus can readily

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utilize GOs in the byproduct waste stream as fermentative substrates to produce value-added beverages, biofuels and biochemicals (Solaiman et al., 2006 & 2007; Karp et al., 2016; Cheng et

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al., 2017; Benedetti et al., 2016; Chua et al., 2017; Jung et al., 2010; Salari et al., 2019). Most microorganisms in the genus Pseudomonas however, do not possess the α-Gal needed to cleave

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the galactose(s) off the GO molecules (Katrolia et al. 2014). Many of these pseudomonads (Poblete-Castro et al., 2012) can produce commercially interesting microbial bioproducts

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including biodegradable polymers (e.g., polyhydroxyalkanoates, PHA) (Preusting et al., 1990; Solaiman and Ashby 2005) and biosurfactants (e.g., rhamnolipids, RL) (Jarvis and Johnson

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1949; Gunther et al. 2005). In order to take advantage of the GO-containing waste streams as low-cost feedstocks, it is therefore desirable to genetically engineer these microorganisms to

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express α-Gal enzyme activity (Suzuki et al., 1969; Ramalingam et al., 2007; Viana et al. 2006). We previously reported the cloning and expression of Streptomyces coelicolor α-galactosidase

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(α-gal) gene (Kondoh et al. 2005) in P. chlororaphis (Solaiman et al. 2018). P. chlororaphis was the target microorganism for our study because it is versatilely capable of producing PHA and

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RL (Solaiman et al. 2006). The recombinant P. chlororaphis strains expressing α-Gal enzyme activity however, only appreciably hydrolyze GO (i.e., raffinose) when the cells are first

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permeabilized by an incubation with ethylenediaminetetraacetic acid (EDTA) (Solaiman et al. 2018). The present study is a continuation of the previous work (Solaiman et al. 2018), wherein

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we alleviated the EDTA-pretreatment step by modifying the bioprocess protocol developed previously (Solaiman et al. 2018) and by attempting to genetically fusing a secretion signal

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sequence to the α-Gal enzyme. We also employed industrially supplied SM and TW to evaluate the feasibility of these new approaches. The main objective of the study is to develop a

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potentially economically-practical process using genetically engineered P. chlororaphis capable of utilizing low-cost SM and TW agro-industrial byproducts as feedstock.

108 Material and methods 110 Microorganisms, culture media, and plasmids 112 P. chlororaphis NRRL B-30761 (Gunther et al. 2007) was from ARS Culture Collection (Peoria, 114

IL). Various strains of E. coli used as host organisms in molecular cloning experiments were

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purchased from New England Biolabs (Ipswich, MA), Invitrogen (Carlsbad, CA), or Clonetech 116

Laboratories (Mountain View, CA). P. chlororaphis transformants containing an expression vector pBS-dAG (designated previously as P. chlororaphis [pBS-dAG]) and a chromosomally

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integrated α-gal gene (P. chlororaphis [chr::AG]) had been described previously (Solaiman et al. 2018). Bacterial cultures were routinely grown in LB medium (1% w/v tryptone, 0.5% w/v yeast

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extract, 0.5% NaCl) at 30 °C (for P. chlororaphis) or 37 °C (for E. coli) with shaking (200-250 rpm), and supplemented with tetracycline (12 µg ml-1) or gentamicin (35 µg ml-1) as appropriate.

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For the studies of GO degradation by P. chlororaphis strains, a Medium E* (Brandl et al., 1988) (per liter: (NH4)2HPO4, 1.1 g; K2HPO4, 5.8 g; KH2PO4, 3.7 g; 1M MgSO4 stock solution, 1ml;

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Microelement Solution, 1 ml) supplemented with a specified amount of GO-substrate (described in detail in a following section) and antibiotic (if necessary) was used. (Composition of

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Microelement Solution (per liter): FeSO4·7H2O, 2.8 g; MnCl2·4H2O, 2 g; CuCl2·2H2O, 0.17 g; ZnSO4·7H2O, 0.3 g; CaCl2·2H2O, 1.67 g; CoSO4·7H2O, 2.9 g.) Plasmid vector pBS-dAG has

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been described (Solaiman et al. 2018).

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Molecular cloning procedures

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All routine molecular-biology procedures were carried out according to protocols described in Ausubel et al. (1987). Enzymes needed for cloning work were purchased from commercial

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sources. Plasmid DNAs were isolated from bacterial transformants using a GenElute Plasmid Prep Kit (Sigma-Aldrich, St. Louis, MO). Synthetic DNA fragments TliAC3 (346 bps) and

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PrtAC3 (328 bps) (Eom et al. 2016) were ordered from GENEWIZ (South Plainfield, NJ). Oligonucleotides used as PCR primers were designed using Clontech Laboratories’s In-Fusion

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Primers Design web-based program and were purchased from Sigma-Aldrich. Transformation of P. chlororaphis was accomplished by an electroporation method (Solaiman & Swingle, 2010).

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Nucleic acid sequence confirmation was carried out using an Applied Biosystems 3730 DNA Analyzer (Life Technologies Corp, Carlsbad, CA).

142 Characterization by recombinant strains in a 2-stage condensed-cell-density bioprocess 144 All cultures in the following experiments were grown in a shaker/incubator set at 30°C and 200 146

rpm rotary shaking. Overnight (16-18 h) seed cultures of P. chlororaphis were prepared in LB media (2 ml) containing an appropriate antibiotic. The seed cultures (2 ml) were used to

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inoculate 50 ml of LB media containing the appropriate antibiotic in 125-ml Erlenmeyer flasks. After 16-40 h (i.e., 1-2 overnights) growth, the 50-ml cultures were used to inoculate 400 ml of

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LB media with the appropriate antibiotic in 1-L Erlenmeyer flasks. Following a 1-2 overnight growth, cells from the 400-ml cultures were harvested aseptically by centrifugation (4°C, 5,400 x

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g, 20 min) for use as inoculum in the subsequent condensed-cell-density bioprocess. To this end, the harvested cells were resuspended in E* media (200-ml volume in 500-ml Erlenmeyer flasks)

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containing an appropriate antibiotic and further supplemented with one of the following carbon sources depending on the experiments: 1) 0.5% (w/v) of raffinose (Sigma-Aldrich, St. Louis,

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MO); 2) 0.22% (w/v) of stachyose (Sigma-Aldrich); 3) 5% (v/v) of a crude soy molasses sample (Central Soya, Gibson City, IL); or 4) 50% (v/v) of a tofu whey sample obtained from a

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manufacturer of soy-based foods in Philadelphia, PA. Aliquots (5-10 ml) of the cultures were removed at the specified times and, after recording the absorbance (at 1/10-dilution) at 600 nm

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(A600nm), were stored in a -20°C freezer for subsequent analysis by high-pressure-liquid-

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chromatography (HPLC) method. Aliquots of cultures were also sampled at the end of 162

fermentation and the biomass values (g/L) were calculated by using the weight of the cell pellets obtained after centrifugation (4°C, 12,000 x g, 5 min).

164 HPLC analysis to determine GO consumption 166

GO consumption by cells in the condensed-cell-density cultures was determined through HPLC monitoring of the decrease of the sugar contents. HPLC analysis was carried out essentially as

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described previously (Solaiman et al. 2015). Briefly, culture samples were centrifuged (room temperature, 12,000 x g, 2-10 min as needed), and the supernatant was filtered on a Whatman

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UniPrep Syringeless Filter unit (0.2 µm PVDF membrane). The filtered samples were analyzed on an HPLC-ELSD (evaporative light scattering detector) equipped with an ES Chromegabond

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Carbohydrate Column (ES Industries; 25cm X 4.6mm; 5µ 100Å). Sample (10-20 µl) was injected into the column. A gradient mobile phase consisting of water (A), methanol (B), and

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acetonitrile (C) was used at a flow rate of 1 ml/min. The mobile phase elution steps were as follows: 0-12 min, 25/75, A/C; 12-17 min, 25/75, A/C → 25/15/60, A/B/C; 17-25 min, 25/15/60,

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A/B/C; 25-28 min, 25/15/60, A/B/C → 25/75, A/C; 28-31 min, 25/75, A/C. The ELSD unit was set at 40°C with an N2 gas flow-rate of 1 ml/min.

178 Results 180 Construction of P. chlororaphis containing secretion signal-gal fusion genes 182

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The secretion signals of a lipase enzyme (TliAC3; 346 bps) and of a metalloprotease A enzyme 184

(PrtAC3; 328 bps) of Pseudomonas fluorescens were tested in this study (Ahn et al. 1999; Eom et al. 2016). Figure 1 shows the schematic drawing of the expression plasmids in which the

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secretion signal fragments TliAC3 and PrtAC3 were respectively spliced into the C-terminus of the S. coelicolor α-galactosidase catalytic fragment (dAG) (Kondoh et al. 2005). A brief

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description for the construction of these expression plasmids follows: The TliAC3 and PrtAC3 gene fragments (GENEWIZ, South Plainfield, NJ) were amplified with primer-pair TliAC3-

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F/TliAC3-R and PrtAC3-F/PrtAC3-R (Supplemental Table S1), respectively, to render their termini compatible for insertion into a linearized pBS-dAG vector (see following description) by

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using an in-fusion method with an In-Fusion Kit (Takara Bio USA, Mountain View, CA). Separately, pBS-dAG (ca. 9 kb) was linearized by performing an inverse-PCR reaction using a

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LongRange PCR Kit (Qiagen, Valencia, CA) with primer-pair DS19_47VecFW-A/ DS19_47VecRV-B (Supplemental Table S1). Figure 1A shows the annealing sites of these

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primers on the vector plasmid. In-fusion cloning of TliAC3 and PrtAC3 into the linearized pBS29-dAG was accomplished using primer-pairs DS19_47TliInFsnF/DS19_47TliInFsnR and

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DS19_47PrtInFsnF/DS19_47PrtInFsnR, respectively, in an In-Fusion reaction performed according to the kit manufacturer’s protocol (In-Fusion Kit, Takara Bio USA). The in-fusion

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reaction mixtures were used directly to transform E. coli, and the recombinant plasmids were isolated and verified by restriction enzyme analysis. The nucleotide sequences of the resulting

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fusion genes in the recombinant plasmids were determined to verify the correct in-frame fusion of the secretion signal fragment to the α-galactosidase gene catalytic fragment (dAG). P.

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chlororaphis was transformed by electroporation with the recombinant plasmids pBS-dAG-

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TliAC3 and pBS-dAG-PrtAC3 (Figure 1B) to yield, respectively, P. chlororaphis strains [dAG206

TliAC3] and [dAG-PrtAC3] (Supplemental Table S1).

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Biomass yields of the recombinants in condensed-cell-density bioprocess

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We evaluated the cell viability of the recombinant strains in the 2-stage condensed-cell-density processes by measuring the final biomass yields in various GO-containing media at the

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completion of the second-stage 7-day incubation. A substantial decrease in biomass yield would indicate the occurrence of unacceptably extensive cell lysis due to the lack of cell viability (i.e.,

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cell death). The biomass yields of recombinant strains cultured in LB medium for 1-2 days were used for comparison. Table 1 shows that in LB medium, all strains showed biomass density

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values of 11.0 – 12.5 g/l medium. In E* media supplemented either with raffinose or stachyose, very similar values of biomass density in the range of 10.0 – 13.0 g cell mass/L culture were

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observed except for P. chlororaphis [dAG-TliAC3] that yielded a lower value of 8.5 g/L (Table 1). In E*+tofu whey complex medium, the observed biomass yields were also in the similar

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range of 10.8 – 14.2 g/L for all strains tested (Table 1). In E*+soy molasses complex medium, however, noticeably higher biomass yields of 18.5 – 20.5 g/L were recorded. This could be

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attributed to the insoluble residues associated with the crude feedstock. Overall, these results suggested that the various genetic modification on P. chlororaphis did not adversely affect the

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biomass density of the strains after a 7-day incubation in GO-containing E* media.

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Consumption of GO in defined media

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The GO-hydrolyzing activity of the recombinant strains was first assessed in the 2-stage condensed-cell-density process using raffinose and stachyose as a carbon source in an E*

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medium (Brandl et al. 1988). We had previously showed the necessity to permeabilize the cells by treatment with EDTA to observe GO hydrolyzing activity by P. chlororaphis recombinants

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(Solaiman et al. 2018). In the present study, our main goal was to avoid this extra step of EDTAtreatment of the cells. We started by first comparing the consumption of raffinose by [dAG]

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strain versus the two newly constructed strains that contain the secretion signal fragments TliAC3 and PrtAC3, respectively. Figure 2A shows the consumption of raffinose by these

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strains. The results showed that the 2-stage condensed-cell-density process successfully detected the raffinose-hydrolyzing activity of P. chlororaphis [dAG] without the necessity to pre-treat the

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cells with EDTA (Fig. 2A). The initial concentration of raffinose (0.5%, w/v) in the medium was reduced to 0.3% (w/v) in 3 days and to <0.1% (w/v) in 7 days (Fig. 2A, solid black bars). This is

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a significant improvement of the process in comparison to the previous study in which an extra step of cell permeabilization by EDTA was necessary (Solaiman et al. 2018). Unexpectedly,

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however, the results with the newly constructed [dAG-TliAC3] and [dAG-PrtAC3] strains containing the secretion signal fragments fused to dAG enzyme did not show significant

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reduction of raffinose in the media. It appeared that the catalytic activity of the fusion enzymes dAG-TliAC3 (530 amino-acid) and dAG-PrtAC3 (533 amino-acid) had been adversely affected

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by the in-frame-fused secretion signal fragments. We next examined stachyose hydrolyzing activity of these three test strains in the 2-stage

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condensed-cell-density process. Again, unlike previous study in which cell permeabilization with EDTA was required, results in Figure 2B (solid black bars) showed that the [dAG] strain was

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able to reduce the stachyose concentration from the initial level of 0.21% (w/v) to 0.15% (w/v) in

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7 days using the 2-stage process. It is interesting to observe that unlike the raffinose substrate, 252

stachyose concentration in the cultures of [dAG-TliAC3] (Fig. 2B, gray bars) and [dAG-PrtAC3] (Fig. 2B, hatched bars) was reduced after the 7-day incubation. Furthermore, the extent of the

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decrease of stachyose concentration for both the [dAG-TliAC3] (from initial 0.23%, w/v to final 0.17%, w/v) and [dAG-PrtAC3] (from 0.22%, w/v to 0.13%, w/v) cultures was like that of

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[dAG]. This decrease of stachyose level in the three cultures was likely a non-enzymatic event such as adsorption to cells or to dead-cell debris.

258 Effect of nitrogen source concentration on GO consumption 260

The validation of the 2-stage condensed-cell-density process to express raffinose-hydrolyzing activity of P. chlororaphis [dAG] (Fig. 2A, solid black bars) without the extra step of

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permeabilizing the cells with EDTA prompted us to re-study the relative GO-hydrolyzing activities of P. chlororaphis wild-type (WT; NRRL B-30176), [dAG] and [chr::AG] strains.

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Figure 3A shows the raffinose consumption patterns by the 3 (three) test strains in the 2-stage process over a 7-day incubation in an E* medium (Brandl et al. 1988). As with the results of the

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previous study (Solaiman et al., 2018), the [dAG] strain exhibited the highest raffinosehydrolyzing activity among the 3 test strains, leading to a near-complete consumption of

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raffinose in the culture medium in 7 days (Fig. 3A, dAG group). The [chr::AG] strain also showed a significant raffinose-hydrolyzing activity, decreasing the concentration of raffinose

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from the initial 0.5% (w/v) to 0.31% (w/v) in 7 days (Fig. 3A, chr::AG group). As expected however, the WT strain did not exhibit raffinose-hydrolyzing activity (Fig. 3A, Wild type

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group).

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Since the E* medium was developed as a nitrogen-limited medium (1.1 g (NH4)2HPO4/l 274

medium; Brandl et al. 1988) to induce PHA biosynthesis in Pseudomonads, there is a need to investigate the effect of nitrogen-source concentration on raffinose-hydrolyzing activity in the

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test strains. Accordingly, we formulated a “high-nitrogen-source” E* medium by using 6-time higher concentration of the nitrogen-source (i.e., 6.6 g (NH4)2HPO4/L medium) than that in the

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original E* medium. Figure 3B shows the raffinose consumption patterns of the 3 (three) test strains in the high-nitrogen-source E* medium over a 7-day period. The [dAG] strain was still

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the most active organism among the test strains in hydrolysing the raffinose in the medium (Fig. 3B). Comparison of the respective raffinose hydrolysing rates of [dAG] and of [chr::AG] in the

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two E* media showed noticeably higher rates in the limited-nitrogen-source E* (Fig. 3A) than in the high-nitrogen-source medium (Fig. 3B). On day-3 in limited-nitrogen-source E*, [dAG]

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culture had reduced the initial raffinose concentration of 0.45% (w/v) to 0.2% (w/v) (Fig. 3A, dAG group); and the [chr::AG], from 0.5% (w/v) to 0.4% (w/v) (Fig. 3A, chr::AG group). In the

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high-nitrogen-source E* medium at day-3, however, [dAG] only reduced the initial raffinose concentration of 0.47% (w/v) to 0.33% (w/v) (Fig. 3B, dAG group); and [chr::AG] had hardly

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reduced the original amount of raffinose (Fig. 3B, chr::AG group). The wild-type strain remained inactive in hydrolysing raffinose in this experiment (Fig. 3B, Wild type group).

290 Consumption of GO in agro-industrial waste media 292 Based on the information gathered from preceding studies using chemically defined GO 294

substrates (i.e., raffinose and stachyose), we proceeded to test the GO-utilizing activities of these strains in E* medium containing complex GO source, i.e., soy molasses (SM) and tofu whey

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(TW). The description and characterization of soy molasses used in this study had been previously described (Solaiman et al. 2007). In the present study, we had determined the

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concentrations of the major soybean sugars (i.e., sucrose, raffinose, and stachyose) in 4 (four) tofu whey samples collected over a 2-mo. period at a local soy-based foods manufacturer; these

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results are shown in Table S2. For the study using SM, the incubation medium E* was supplemented with 5% (v/v) soy

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molasses to achieve a total soy sugars concentration of ca. 1.6% (w/v) (Solaiman et al. 2007). Based on Table S2, the E*+SM therefore contained 0.1% w/v raffinose and 0.55% w/v

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stachyose. Figure 4 shows the raffinose (Fig. 4A) and stachyose (Fig. 4B) consumption in medium E*+SM over a 7-day incubation period with the 3 (three) test strains. The most active

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strain among the three tested organisms is the [dAG]. It reduced the amount of raffinose in the medium from the original 0.1% w/v to 0.05% w/v and 0.03% in 3 and 7 days, respectively (Fig.

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4A, solid black bars). The [chr::AG] strain, on the other hand, reduced the raffinose in the medium from the initial 0.1% w/v to 0.07% w/v and 0.06% w/v in 3- and 7-day periods,

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respectively (Fig. 4A, gray bars). The wild-type strain did not show raffinose reduction activity in soy molasses medium (Fig. 4A, hatched bars). Figure 4B presented stachyose consumption

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data by the 3 (three) test strains in E*+SM medium. The [dAG] strain reduced the stachyose content from the initial 0.56% w/v to 0.35% w/v and 0.25% w/v in 3 and 7 days of incubation,

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respectively (Fig. 4B, solid black bars). The [chr::AG] strain reduced the stachyose content in a similar rate, decreasing the initial concentration of 0.56% w/v to 0.31% w/v and 0.27% w/v in 3-

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and 7-day incubation, respectively (Fig. 4B, gray bars). The WT strain did not appreciably decrease the stachyose content of the medium; the 3- and 7-day cultures still contained 0.45%

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w/v and 0.43% w/v stachyose, respectively, in comparison to the initial concentration of 0.54% w/v.

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For the study using TW, the incubation medium E* was supplemented by 50% v/v of TW. Based on the data in Table S2, the initial concentrations of raffinose and stachyose in the

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E*+TW medium were calculated as 0.11% w/v and 0.27% w/v, respectively. Figure 5 shows the pattern of raffinose and stachyose consumption by the three test strains after 0 (i.e., initial

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cultures), 2 and 8 days of incubation. The initial levels of raffinose (0.11 ± 0.01% w/v; Fig. 5A) and stachyose (0.27 ± 0.01% w/v; Fig. 5B) were respectively reduced within 2 days to 0.05%

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w/v (for raffinose) and 0.08% w/v (for stachyose) by the [dAG] strain (solid black bars in Figs. 5A and 5B), and remained at these levels to 8 days of incubation. The other two test strains, i.e.,

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[chr::AG] (gray bars in Figs. 5A and 5B) and the wild-type (hatched bars in Figs. 5A and 5B), did not reduce the amounts of raffinose and stachyose in the media up to the 8th day of

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incubation.

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Discussion

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The use of agro industrial waste streams as fermentative feedstocks is an ongoing research emphasis area in biorefineries (Hussain et al., 2019). In this study, we were interested in the soy

336

molasses and tofu whey waste streams generated from soybean processing or utilization industries. The carbon source in these waste streams are the GOs (i.e., raffinose and stachyose)

338

and sucrose. While many Pseudomonads can utilize sucrose, they lack the key enzyme (i.e., αgalactosidase) to break down GOs. We previously reported the genetic modification of P.

340

chlororaphis to express α-galactosidase activity and demonstrated the ability of the recombinants

15

[Type here]

to degrade GOs when the cells were first permeabilized through pretreatment with 342

ethylenediaminetetraacetate (EDTA) (Solaiman et al., 2017). In this study, we successfully demonstrated a two-stage condensed-cell-density bioprocess to enable the use of intact P.

344

chlororaphis transformants to hydrolyze GOs without the need for the extra step of EDTA pretreatment (Figs. 1-5). The cost saving for this move is without saying. The biomass study

346

showed that the total cell-weights (in g/L) in the condensed-cell-density process did not lose weight, suggesting that there was no appreciable lysis of the bacterial cells (Table 1). This is

348

important because the stability and intactness of the whole cells (i.e., the biocatalyst) is required for the functioning of the process. Two-stage fermentation approach is commonly practiced in

350

bio-processing research and industry. Daniel et al. (1999) had implemented this process for highyield production of sophorolipid biosurfactant by Cryptococcus curvatus and Candida bombicola

352

using cheese whey concentrates as substrates. Zhou et al. (2018) employed a two-stage fermentation

process for the integrative production of gluconic acid and xylonic acid by Gluconobacter 354

oxydans using corn stover as feedstock. We had utilized a similar two-stage bioprocess to produce block-copolymeric short-chain poly(hydroxyalkanoates) from Burkholderia sacchari

356

using xylose and levulinic acid as substrates (Ashby et al., 2018). The present results further attested to the versatility of this approach in bioprocessing, allowing the genetically engineered

358

P. chlororaphis to degrade GOs without the added cost of the pretreatment step of permeabilization of the cells.

360

The overall medium composition is an important factor affecting the GO-hydrolyzing capability of the bacteria. Results in Fig. 3 showed that for the [dAG], a low (limited) nitrogen

362

content (Fig. 3A) favoured the consumption of raffinose than a high nitrogen content (Fig. 3B). For the [chr::AG] strain, however, the high nitrogen content (Fig. 3B) promoted a more

16

[Type here]

364

extensive reduction of raffinose level at day-7 (Fig. 3B, hatched bars) than the low nitrogen content environment (Fig. 3A, hatched bars). Comparison of Fig. 4 and Fig. 5 also revealed that

366

the [dAG] and [chr::AG] exhibited different GO-hydrolyzing patterns in soy molasses versus in tofu whey media. In soy molasses, it was found that both the [dAG] and the [chr::AG] strains

368

were capable of hydrolysing raffinose and stachyose (Fig. 4). In tofu whey medium, however, only [dAG] was capable of hydrolysing stachyose (Fig. 5B). The explanation(s) for the different

370

patterns of GO-hydrolyzing activities of [dAG] and [chr::AG] when incubated in different media are complex and require further research to delineate. This observation nevertheless cautions the

372

necessity of a holistic design of a system in its entirety and on a case-by-case manner in order to achieve the desired performance of a bioprocess. Other researchers have presented different

374

bioprocesses when using different agro-industrial waste streams as feedstocks to produce different types of end products. For examples, Salari et al. (2019) evaluated the production of

376

bacterial cellulose by Gluconacetobacter xylinus in sugar beet molasses, cheese whey and standard Hestrin–Schramm (HS) media; Jung et al. (2010) characterized the synthesis of

378

cellulose by an Acetobacter sp. V6 in molasses and corn steep liquor medium; Chua et al. (2017) described the fermentation of soy whey into soy-based alcoholic beverage by various

380

Saccharomyces cerevisiae strains; Karp et al. (2017) demonstrated the production of bioethanol fuel through fermentation of soy molasses by Saccharomyces cerevisiae or Zymomonas mobilis

382

to achieve an ethanol yield as high as 163 L per ton of dry molasses at pilot scale trial. In the same manner, future study is thus required to fine-tune the various combinations of [dAG],

384

[chr::AG], soy molasses and tofu whey to enable the synthesis of the bioproducts of the P. chlororaphis recombinant strains, i.e., the PHA and RL.

17

[Type here]

386

When fused to a recombinant enzyme, a protein secretion signal could potentially promote the extracellular re-localization of the enzyme, therefore improving the accessibility of the enzyme

388

to its substrate in the culture medium. Ahn et al. (1999) first reported the identification of a potential extracellular secretion targeting sequence in the C-terminal portion of the lipase

390

enzyme (tliA) of Pseudomonas fluorescens. Eom et al. (2016) subsequently found a secretion targeting sequence in the C-terminal portion of the metalloprotease A enzyme (prtA) of P.

392

fluorescens. Furthermore, Eom et al. (2016) demonstrated that only a short fragment of the Cterminal secretion sequences of tliA (tliAC3 fragment, 350 base-pair) and prtA (prtAC3 fragment,

394

330 base-pair) was required to direct the secretion of a heterologous mannase in P. putida, E. coli and S. marcesens. In this study we genetically fused the two P. fluorescens secretion signal

396

fragments, i.e., tliA and prtA, respectively, to the recombinant α-Gal in P. chlororaphis. We then assessed the effectiveness of this approach to increase the hydrolysis of GOs in the culture

398

medium. The results showed not only a lack of improvement but a detrimental effect of the fusion constructs on the GO hydrolysis activities of the cells (Fig. 2). Since earlier studies by

400

others had established the general effectiveness of TliAC3 and PrtAC3 to promote the extracellular transport of the fused enzyme including a lipase (Ahn et al. 1999), a

402

metalloprotease (Eom et al. 2014), and endo-β-1,4-mannanase (Eom et al. 2016), the results of the present study are highly suggestive of the creation of catalytically inactive fusion dAG-

404

TliAC3 and dAG-PrtAC3 enzymes. Future studies would entail the testing of many other extracellular secretion targeting sequences such as pelB and ompA (Zhu et al., 2016) and the

406

1,175 bacterial signal peptide sequences described by Owji and Hemmati (2018).

408

Conclusions

18

[Type here]

We have genetically engineered P. chlororaphis to express α-galactosidase activity via plasmid 410

vector ([dAG] strain) and chromosomal integration ([chr::AG] strain), and have also experimented with the use of secretion signals (TliAC3 and PrtAC3) to try to increase enzyme

412

secretion. We then compared the genetically modified strains in a condensed-cell-density bioprocess for their ability to consume soy sugars, i.e., raffinose and stachyose. We found that

414

[dAG] and [chr:AG] strains consumed raffinose but not stachyose. The use of secretion signals unexpectedly resulted in in-frame fusion proteins lacking α-galactosidase activity. We further

416

tested [dAG] and [chr::AG] in complex culture media containing industrial soy molasses and tofu whey, and found that both strains were capable of reducing the soy sugars in the media. The

418

results of the study thus laid down valuable knowledge-base for future work to develop P. chlororaphis and other important pseudomonads to metabolize GO-containing fermentation

420

feedstocks to support cost-effective production of bioproducts.

19

[Type here]

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540

542

544 Figure legends 546

Fig. 1. Schematics of plasmid maps. A. Plasmid map of pBS-dAG. B. Plasmid maps of pBS-dAG-PrtAC3 (left) and pBS-dAG-TliAC3. Abbrev.: TcR, tetracycline-resistance

548

determinant; rep, replication protein; dAG, gene coding for the N-terminal catalytic domain of S. coelicolor α-galactosidase; P2, promoter sequence from P. syringae; KmR, kanamycin-

550

resistance determinant. Fig. 2. Galacto-oligosaccharide (GO) hydrolyzing activity of P. chlororaphis

552

expressing dAG-TliAC3 and dAG-PrtAC3 fusion enzymes. Cells (4-5 g) from cultures grown in rich LB medium (400 ml) were harvested by centrifugation and inoculated into E*

554

media (200 ml) containing the specified GO. Cultures were sampled (4-5 ml; stored in a -20°C 25

[Type here]

freezer) at intervals. GO contents were determined by HPLC. A. Incubation in E* + raffinose 556

(0.5%, w/v).

, [dAG];

stachyose (0.22%, w/v). 558

, [dAG-TliAC3]; , [Wild-type];

, [dAG-PrtAC3]. B. Incubation in E* +

, [dAG-TliAC3];

, [dAG].

Fig. 3. Effect of nitrogen-source concentration on raffinose-hydrolyzing activity of P. chlororaphis strains. A. E* medium containing 1.1 g/L (NH4)2HPO4. B. E* medium

560

containing 6.6 g/L (NH4)2HPO4. Symbols:

, Day-0;

, Day-3;

, Day-7.

Fig. 4. GO-hydrolyzing activity of P. chlororaphis strains in soy molasses. The E* 562

medium was supplemented with crude soy molasses (5%; v/v) as carbon source. A. Consumption of the raffinose component in soy molasses. B. Consumption of the stachyose component in soy

564

molasses. Symbols:

, [dAG];

, chr::AG;

, Wild-type.

Fig. 5. GO-hydrolyzing activity of P. chlororaphis strains in tofu whey. The E* 566

medium was supplemented with samples of tofu whey (50%; v/v) as carbon source. A. Consumption of the raffinose component in tofu whey. B. Consumption of the stachyose

568

component in tofu whey. Symbols:

, [dAG];

570

26

, chr::AG;

, Wild-type.

[Type here]

Figure 1

A.

B.

572

27

[Type here]

Figure 2 A. Raffinose Consumption

B. Stachyose Consumption

574

28

[Type here]

Figure 3 A. Raffinose Consumption in E* Medium (Limited Nitrogen Source)

B. Raffinose Consumption in E* Medium (High Nitrogen Source)

576

29

[Type here]

Figure 4 A. Raffinose Consumption in E* Medium Containing Soy Molasses

B. Stachyose Consumption in E* Medium Containing Soy Molasses

578

30

[Type here]

580

Figure 5 A. Raffinose Consumption in E* Medium Containing Tofu Whey

582 584 586 588 590 592 594 596 598 600 602 604

B. Stachyose Consumption in E* Medium Containing Tofu Whey

606 608

31

[Type here]

610

Table 1. Cell biomass of P. chlororaphis strains in various culture media

Culture Medium2

2

Wild type

[pBS-dAG]

[chr::AG]

[pBS-dAGTliAC3]

[pBS-dAGPrtAC3]

LB Medium

12.5

11.3

11.3

11.0

11.0

E* + Raffinose

11.5

11.5

11.0

10.5

11.5

E* + Stachyose

13.0

10.0

--

8.5

--

18.5

19.0

20.5

--

--

14.2

10.8

11.7

--

--

E* + Soy Molasses E* + Tofu Whey 1

Cell biomass1 of P. chlororaphis strain (g/L)

Weight of wet cell pellet collected at completion of the experiments Total volume of medium was 200 ml, except E* + Tofu Whey (100 ml)

612

32

[Type here]

Table S1. Bacterial Strains and Primers Primers Name

Sequence (5′

3′)

TliAC3-F

AATCTAGAGGCAGCGACGGCAATGACCTG

TliAC3-R

GGAAAGCTTCATGAACCGCCGATAATC

Comment

Primer-pair for In-Fusion insertion of TliAC3 into pBS29-P2-dAG

PrtAC3-F PrtAC3-R

GCCRATYCTSTACACCATYCCG Primer-pair for In-Fusion insertion of PrtAC3 into pBS29-P2-dAG CACAGCATAACTGGACTGATTTC

DS19_47VecFW-A

GGCCTCTAGGCCAGATCCAGCGGCATCTGGGT

DS19_47VecRV-B

GTGGCCACCACGAAGCTTATACTGAGCCCATT

DS19_47PrtInFsnF

CTTCGTGGTGGCCACGGCGGTGGCGGTGCCGAC

DS19_47PrtInFsnR

TCTGGCCTAGAGGCCTGCATCACGCCACGATGT

DS19_47TliInFsnF

CTTCGTGGTGGCCACGGCAGCGACGGCAATGAC

DS19_47TliInFsnR

TCTGGCCTAGAGGCCCATGAACCGCCGATAATCC

Primer-pair to amplify and linearize pBS29-P2-dAG

Primer-pair to perform In-Fusion annealing reaction between linearized pBS29P2-dAG and the PrtAC3 insert Primer-pair to perform In-Fusion annealing reaction between linearized pBS29P2-dAG and the TliAC3 insert

Bacteria Strains

Description

P. chlororaphis NRRL B-30761

Wild-type strain; synthesize rhamnolipid

P. chlororaphis [chr::AG] P. chlororaphis [pBSdAG] P. chlororaphis [pBSdAG-TliAC3] P. chlororaphis [pBSdAG-PrtAC3]

Reference Gunther et al. 2007 (Patent 7,202,063)

Chromosomally integrated S. coelicolor α-gal gene; gentamycin resistant Containing recombinant plasmid pBS-dAG; tetracycline resistant Containing recombinant plasmid pBS-dAG-TliAC3; tetracycline resistant Containing recombinant plasmid pBS-dAG-PrtAC3; tetracycline resistant

33

Solaiman et al. 2018 (bab) Solaiman and Swingle 2010 This study This study

[Type here]

34

[Type here]

Table S2. Soy Sugars Contents in SM and TW Feedstock

Raffinose

Stachyose

1

2% w/v

11% w/v

2

0.21% w/v

0.54% w/v

SM

TW 2

4

1

Data for soy molasses (SM) were from Montelongo et al. (1993)

2

Values for tofu whey (TW) were determined by HPLC-ELSD analysis in the present study. TW

samples were diluted in 1:1 (v/v) ratio with Milli-Q water; filtered on Whatman Mini-UniPrep 6

Syringeless Filter units (.2 µ PVDF membrane; GE Healthcare Bio-Sciences, Pittsburgh, PA); and analyzed on a Shimadzu HPLC-ELSD instrument using column and eluent conditions as

8

described in the Materials and Methods section. Calibration curves were established using serially increasing volumes of standard solutions of raffinose (0.5% w/v) and stachyose (0.5%

10

w/v) in HPLC analysis. S.D.’s (n=4) of the calculated values were < ±7.5%.

35

[Type here]

Supplemental Figure S1

2

4

6

8

10

12

14

Schematic of Soy Sugar Hydrolysis. Enzymes required for the process are α-galactosidase (α-

D-galactoside galactohydrolase, EC 3.2.1.22) and invertase (β-fructofuranosidase, EC 3.2.1.26). 16

Abbreviations: α-D-gal, α-D-galactose; α-D-glu, α-D-glucose; β-D-fru, β-D-fructose.

18

20

36

Table 1. Cell biomass of P. chlororaphis strains in various culture media

Culture Medium2

Cell biomass1 of P. chlororaphis strain (g/L) Wild type

[pBS-dAG]

[chr::AG]

[pBS-dAGTliAC3]

[pBS-dAGPrtAC3]

LB Medium

12.5

11.3

11.3

11.0

11.0

E* + Raffinose

11.5

11.5

11.0

10.5

11.5

E* + Stachyose

13.0

10.0

--

8.5

--

E* + Soy Molasses

18.5

19.0

20.5

--

--

E* + Tofu Whey

14.2

10.8

11.7

--

--

1 2

Weight of wet cell pellet collected at completion of the experiments Total volume of medium was 200 ml, except E* + Tofu Whey (100 ml)