De novo Microbial Biosynthesis of a Lactate Ester Platform

Green organic solvents such as lactate esters have broad industrial applications. Manufacturing and use of these biodegradable solvents from renewable feedstocks help benefit the environment. This study presents a microbial conversion platform for direct fermentative biosynthesis of lactate esters from fermentable sugars. To enable the de novo microbial biosynthesis of lactate esters, we first designed a sugar-to-lactate ester module, consisting of a lactate dehydrogenase (ldhA), a propionate CoA-transferase (pct), and an alcohol acyltransferase (AAT) to convert sugar to lactate esters. By generating a library of five sugar-to-lactate ester modules with divergent AATs, we screened for the best module(s) capable of producing a wide range of linear, branched, and aromatic lactate esters with an external alcohol supply. By co-introducing a sugar-to-lactate ester module and an alcohol (i.e., ethanol, isobutanol) module into a modular Escherichia coli (chassis) cell, we demonstrated for the first time the de novo microbial biosynthesis of ethyl and isobutyl lactate esters directly from glucose. In an attempt to enhance ethyl lactate production as a proof-of-study, we re-modularized the pathway into the upstream module to generate the ethanol and lactate precursors and the downstream module to generate lactyl-CoA and condense it with ethanol to produce the target ethyl lactate. By manipulating the metabolic fluxes of the upstream and downstream modules though plasmid copy numbers, promoters, ribosome binding sites, and environmental perturbation, we were able to probe and alleviate the metabolic bottlenecks by improving ethyl lactate production by 4.96-fold.


Introduction
Lactate esters are generally produced by esterification of lactic acid with alcohols using 44 homogenous catalysts (i.e., sulfuric acid, hydrogen chloride, and/or phosphoric acid) under high 45 temperature reaction conditions [4]. However, the esterification reactions are thermodynamically 46 unfavorable (G = +5 kcal/mol) in aqueous solutions and often encounter significant challenge 47 due to self-polymerization of lactate [5]. Alternatively, microbial catalysts can be used to produce 48 these esters in a thermodynamically favor reaction (G = -7.5 kcal/mol) in an aqueous phase 49 environment at room temperature and atmospheric pressure [6][7][8][9][10][11][12]. This reaction uses an alcohol 50 acyl transferase to generate an ester by condensing an alcohol and an acyl-CoA. Currently, the de 51 novo microbial biosynthesis of lactate esters directly from fermentable sugars has not yet been 52 demonstrated in microbes. 53 In this work, we aimed to demonstrate the feasibility of microbial production of lactate 54 esters as green organic solvents, from renewable resources. To establish a de novo microbial 55 biosynthesis of a lactate ester platform directly from fermentable sugars, we first started by in vivo 56 First, the TT7 terminator was added between the MCS1 and MCS2 of the pRSFDuet-1 backbone 148 by assembling three DNA fragments: i) the pct gene amplified from pJW001 using the primer pair 149 JW_0013/JW_0032, ii) the linker containing TT7 terminator from pETite* using the primer pair 150 JW_0033/JW_0034, and iii) the backbone from pRSFDuet-1 using the primer pair 151 JW_0017/JW_0018, generating the first intermediate plasmid, pJW023. Then, the original RBS in 152 MCS1 of pJW023 was replaced with synthetic RBSs of various strengths by assembling two DNA 153 fragments: i) the pct gene amplified from pJW001 with the synthetic RBS sequences with 154 predicted translation initiation rates at 90, 9000, or 90000au for pJW024, pJW025 or pJW026 155 using the primer pair JW_0035/JW_0036, JW_0037/JW_0036, or JW_0038/JW_0036, 156 respectively, and ii) the backbone amplified from pJW023 using the primer pair 157 JW_0039/JW_0040 for pJW024, JW_0041/JW_0040 for pJW025, or JW_0042/JW_0040 for 158 pJW026, respectively, generating the second intermediate plasmids, pJW024-026. Lastly, the final 159 nine downstream modules (pJW027-035) were constructed by assembling a combination of two 160 DNA fragments: i) the VAAT gene amplified from pDL006 with the synthetic RBS sequences 161 predicted with translation initiation rates of 90, 9000, or 90000au for pJW027/pJW030/pJW033, 162 pJW028/pJW031/pJW034, or pJW029/pJW032/pJW035 using the primer pair 163 JW_0043/JW_0010, JW_0044/JW_0010, or JW_0045/JW_0010, respectively, and ii) the 164 backbone amplified from pJW024, pJW025, or pJW026 for pJW027-029, pJW030-032, or 165 pJW033-035 using the primer pair JW_0046/JW_0012, JW_0047/JW_0012 or 166 JW_0048/JW_0012, respectively. The RBS Calculator v2.0 [20, 21] was used to generate six 167 synthetic RBS sequences with predicted translation initiation rates of 90, 9000, and 90000au 168 between the PT7 promoter and pct (or VAAT) start codon (Fig. S3) cell density, dual-phase fermentation approach, aerobic cell growth phase followed by anaerobic 195 production phase, was applied. For the first aerobic cell growth phase, the temperature, agitation, 196 and air flow rate were maintained at 37°C, 1000 rpm, and 1 volume/volume/min (vvm) for 4 h, 197 respectively. Then, the oxygen in the medium was purged by sparing nitrogen gas at 2 vvm for 2 198 h to generate anaerobic condition. For the anaerobic production phase, 0.5 mM of IPTG was added 199 to induce the protein expression and the culture temperature and the nitrogen flow rate were 200 maintained at 30°C and 0.2 vvm, respectively. During the fermentation, the pH was maintained at 201 7.0 with 5 M KOH and 40% H3PO4. Bioreactor batch fermentation studies were performed in 202 biological duplicates. 203

Growth inhibition analysis of lactate esters 204
The seed culture of EcDL002 was prepared as described in high-cell density cultures. programmed with an initial temperature of 50°C with a 1°C/min ramp up to 58°C. Next a 25°C/min 244 ramp was deployed to 235°C and then finally held a temperature of 300°C for 2 minutes to elute 245 any residual non-desired analytes. The injection was performed using a splitless mode with an 246 initial injector temperature of 280°C. For the MS system, a selected ion monitoring (SIM) mode 247 was deployed to detect analytes. 248 The SIM parameters for detecting lactate esters were as follows: i) for pentanol, ions 53.00, 249 60.00, and 69.00 detected from 5.00 to 7.70 min, ii) for ethyl lactate, ions 46.00, 47.00, and 75.00 250 detected from 7.70 to 10.10 min, iii) for propyl lactate, ions 59.00, 88.00, and 89.00 detected from 251 10.10 to 11.00 min, iv) for isobutyl lactate, ions 56.00, 57.00, and 59.00 detected from 11.00 to 252 11.60 min, v) for butyl lactate, ions 75.00, 91.00, and 101.00 detected from 11.60 to 12. 30

In vivo screening of efficient AATs critical for lactate ester biosynthesis 265
We began the construction of lactate ester biosynthesis pathways by identifying the best 266 AAT candidate because the substrate specificity of AATs is critical to produce target esters [9]. 267 We designed, constructed, and characterized a library of five sugar-to-lactate ester modules 268 (EcJW101-105) carrying five divergent alcohol acyltransferases including ATF1, ATF2, SAAT, 269 VAAT, and AtfA. For characterization, 2 g/L of ethanol, propanol, butanol, isobutanol, isoamyl 270 alcohol, and benzyl alcohol were added to culture media with 0.5 mM of IPTG for pathway 271 induction to evaluate biosynthesis of six different lactate esters including ethyl lactate, propyl 272 lactate, butyl lactate, isobutyl lactate, isoamyl lactate, and benzyl lactate, respectively, in high-cell 273 density cultures (Fig. 1A). 274 The results show that most of the strains could produce different types of lactate esters with 275 external supply of alcohols ( Fig. 1B, 1C). EcJW104 achieved the highest titer of lactate esters in 276 all cases, producing 1.59 ± 0.04 mg/L of ethyl lactate with a specific productivity of 0.04 ± 0.00 277 mg/gDCW/h in ethanol doping, 5.46 ± 0.25 mg/L of propyl lactate with a specific productivity of 278 0.14 ± 0.01 mg/gDCW/h in propanol doping, 11.75 ± 0.43 mg/L of butyl lactate with a specific 279 productivity of 0.29 ± 0.01 mg/gDCW/h in butanol doping, 9.92 ± 0.08 mg/L of isobutyl lactate 280 with a specific productivity of 0.25 ± 0.00 mg/gDCW/h in isobutanol doping, 24.73 ± 0.58 mg/L 281 of isoamyl lactate with a specific productivity of 0.62 ± 0.01 mg/gDCW/h in isoamyl alcohol 282 doping, and 51.59 ± 2.09 mg/L of benzyl lactate with a specific productivity of 1.91 ± 1.10 283 mg/gDCW/h in benzyl alcohol doping. The lactate ester biosynthesis of EcJW104 exhibited 284 different alcohol substrate preference in the following order: benzyl alcohol > isoamyl alcohol > 285 butanol > isobutanol > propanol > ethanol (Fig. 1B, Supplementary Table S2). 286 Due to the presence of endogenous acetyl-CoA, we also produced acetate esters in addition 287 to lactate esters (Fig. 1) identifying the efficient AAT for lactate ester biosynthesis. We focused on the biosynthesis of 308 ethyl and isobutyl lactate esters. We designed the de novo biosynthesis pathways for ethyl and 309 isobutyl lactate by combining the sugar-to-lactate ester module (pJW005) with the ethanol 310 (pCT24) and isobutanol (pCT13) modules, respectively. By co-transforming pJW005/pCT24 and 311 pJW005/pCT13 into the modular cell EcDL002, we generated the production strains, EcJW201 312 and EcJW202, for evaluating direct conversion of glucose to ethyl and isobutyl lactate esters.  Table S3A). Taken altogether, the direct microbial 324 synthesis of lactate esters from fermentable sugar was successfully demonstrated. Since the lactate 325 ester production was low, the next logical step was to identify and alleviate the key pathway 326 bottlenecks for enhanced lactate ester biosynthesis. As proof-of-principle, we focused on 327 optimization of the ethyl lactate production as presented in the subsequent sections. 328

Identifying and alleviating key bottlenecks of the ethyl lactate biosynthesis pathway 329 a basis to identify potential pathway bottlenecks.
In an attempt to identify the key bottlenecks of 331 the ethyl lactate biosynthesis pathway, we characterized EcJW201 in controlled bioreactors. The 332 results show that EcJW201 produced 9.17 ± 0.12 mg/L of ethyl lactate with a specific productivity 333 of 0.15 ± 0.02 mg/gDCW/h and a yield of 0.19 ± 0.00 mg/g glucose (Fig. 2C Therefore, to enhance ethyl lactate production, it is important to elucidate and alleviate these 344 identified potential bottlenecks. 345

Ethyl lactate exhibited minimal cytotoxicity on cell growth among lactate esters. 346
To determine whether lactate esters inhibited cell growth and hence contributed to low lactate ester 347 production, we cultured the parent strain, EcDL002, in a microplate reader with or without supply 348 of various concentrations of lactate esters including ethyl, propyl, butyl, isobutyl, isoamyl, or 349 benzyl lactate. The results show that ethyl lactate was the least toxic among the six lactate esters 350 characterized where the growth rate (0.47 ± 0.04 1/h) and cell titer (OD = 0.42 ± 0.03) were slightly 351 reduced to 94% and 90%, respectively, upon cell exposure to 5 g/L ethyl lactate. On the other 352 hand, isobutyl lactate was the most toxic among the lactate esters, where cell exposure to only 0.5 353 g/L ester caused 82% and 85% decrease in the growth rate (0.41 ± 0.02 1/h) and OD (0.40 ± 0.03), 354 respectively (Supplementary Figure S2A)

Downstream pathway of the lactate ester biosynthesis is the key bottleneck.
To 363 identify and alleviate the de novo ethyl lactate biosynthesis pathway, we re-modularized it with 364 two new parts: i) the upstream module carrying ldhA, pdc, and adhB for production of lactate and 365 ethanol from sugar and ii) the downstream module carrying pct and VAAT for converting lactate 366 into lactyl-CoA and condensing lactyl-CoA and ethanol (Fig. 3A). We controlled metabolic fluxes 367 of these modules by controlling their plasmid copy numbers and levels of promoter induction with 368 IPTG. By introducing the plasmids pJW007-015 into EcDL002, we generated the strains 369 EcJW106-108 and EcJW203-208, respectively (Fig. 3B). To evaluate the performance of these 370 constructed strains for ethyl lactate production, we characterized them in high cell density cultures 371 induced with various concentrations of IPTG (0.01, 0.1, and 1.0 mM). 372 The results show that EcJW204, carrying the upstream module with a low copy number 373 plasmid (P15A origin) and the downstream module with a high copy number plasmid (RSF1030 374 origin) induced by 0.01 mM of IPTG, achieved the highest titer of ethyl lactate at 11.10 ± 0.58 375 glucose. As compared to EcJW201, EcJW204 achieved 396%, 450%, and 440% improvement in 377 titer, specific productivity, and yield of ethyl lactate, respectively (Fig. 3B, Supplementary Table  378 S5). Interestingly, the best ethyl lactate producer EcJW204 produced the highest titer of lactate 379 and the lowest ethanol production among the characterized nine strains (Fig. 3F and 3G The results show the carbon flux was successfully redistributed from ethanol to lactate, 400 with 83~86% decrease in ethanol production and 67~159% increase in lactate production 401 (Supplementary Table S6A). However, the production of ethyl lactate and ethyl acetate was 402 reduced 87~92% and 92~95%, respectively in all characterized strains as compared to that of 403 EcJW204 (Fig. 4B, Supplementary Table S6B). The low ethyl ester production suggests that a 404 high level of ethanol is critical for VAAT to produce esters. To support this conclusion, we 405  Table S6). Further addition 410 of ethanol up to 10 g/L improved the ethyl lactate and ethyl acetate production by 278~426% and 411 309~592%, respectively (Supplementary Table S6). Interestingly, while the total titer of ethyl 412 esters increased with the increasing addition of ethanol (Fig. 5A), the proportion of ethyl lactate in 413 the total ester slightly increased in the range of 3.2~7.0% (Fig. 5B), suggesting that VAAT prefers 414 acetyl-CoA over lactyl-CoA with ethanol as a co-substrate. Notably, we observed a strong linear 415 correlation between ethyl esters production and the amount of added ethanol (i.e., for ethyl lactate, 416 R 2 = 0.85~0.94; for ethyl acetate, R 2 = 0.99~1.00) (Supplementary Figure S4A). The results 417 suggest abundant availability of ethanol is essential to achieve high production of ethyl esters. A 418 combination of low affinity of VAAT for ethanol and low specificity of VAAT for lactyl-CoA 419 contributed to low ethyl lactate biosynthesis. 420 Pct for conversion of lactate to lactyl-CoA or VAAT for condensation of lactyl-CoA and an alcohol 422 was the most rate limiting step of the downstream module, we redesigned and constructed nine 423 downstream modules (pJW027-035) derived from pJW012 of the best performer EcJW204 using 424 a combination of three synthetic RBSs for Pct expression (synRBSpct#1-3) and three synthetic RBSs 425 for VAAT expression (synRBSVAAT#1-3) (Fig. 4A, Supplementary Figure S3B). We introduced 426 each downstream module into EcDL002 together with the upstream module (pJW007) used in 427 EcJW204 to generate EcJW213-221. We characterized the constructed strains in high cell density 428 cultures induced with 0.01 mM IPTG. 429 The results show that the strains harboring the stronger RBSs for VAAT expression 430 achieved the higher titers of ethyl lactate and ethyl acetate regardless of the RBS strengths for Pct 431 expression (Fig. 4C, Supplementary Table S7). There is a strong linear correlation between ethyl 432 ester production and the strength of RBS for VAAT expression (Supplementary Figure S4B). To 433 further validate these results without the influence of the upstream module, we additionally 434 constructed the strains EcJW109-117 by introducing nine individual downstream modules 435 (pJW027-035) into EcDL002 and then characterized these strains in high cell density cultures with 436 addition of 2 g/L of lactate, 2 g/L of ethanol, and 0.01 mM of IPTG. We could observe the same 437 strong linear correlation between ethyl ester production and high VAAT expression without the 438 upstream module (Fig. 5C) EcJW104, harboring the sugar-to-lactate module with VAAT, produced 6 out of 6 target lactate 460 esters including ethyl, propyl, butyl, isobutyl, isoamyl, and benzyl lactate with the highest titers. 461 Using VAAT, we demonstrated for the first time the direct biosynthesis of ethyl and isobutyl 462 lactate from fermentable sugars. 463 Despite successful demonstration of the de novo biosynthesis of ethyl lactate from glucose, 464 the current production of target esters was relatively low. Through detailed pathway 465 characterization, we found that the inefficient lactate ester pathway flux, not the ester cytotoxicity, 466 hindered ethyl lactate production in the E. coli modular cell. By modularizing the ethyl lactate 467 pathway into upstream and downstream modules and manipulating their metabolic fluxes with 468 various plasmid copy numbers, promoters, and RBSs, we could identify the inefficient downstream 469 module hindered efficient lactate ester biosynthesis. Specifically, VAAT was the most rate limiting 470 step of the downstream module where it has low affinity to ethanol and low specificity to lactyl-471 CoA. While overexpression of the downstream module increased lactate ester biosynthesis by ~5 472 folds, the production level was still low. Future study exploring diversity of AATs and rational 473 protein engineering of these enzymes is warranted for improving product synthesis and expanding 474 a large library of lactate esters to be synthesized.