Microbial production of rhamnolipids: opportunities, challenges and strategies

Abstract Rhamnolipids are a class of biosurfactants which contain rhamnose as the sugar moiety linked to β-hydroxylated fatty acid chains. Rhamnolipids can be widely applied in many industries including petroleum, food, agriculture and bioremediation etc. Pseudomonas aeruginosa is still the most competent producer of rhamnolipids, but its pathogenicity may cause safety and health concerns during large-scale production and applications. Therefore, extensive studies have been carried out to explore safe and economical methods to produce rhamnolipids. Various metabolic engineering efforts have also been applied to either P. aeruginosa for improving its rhamnolipid production and diminishing its pathogenicity, or to other non-pathogenic strains by introducing the key genes for safe production of rhamnolipids. The three key enzymes for rhamnolipid biosynthesis, RhlA, RhlB and RhlC, are found almost exclusively in Pseudomonas sp. and Burkholderia sp., but have been successfully expressed in several non-pathogenic host bacteria to produce rhamnolipids in large scales. The composition of mono- and di-rhamnolipids can also be modified through altering the expression levels of RhlB and RhlC. In addition, cell-free rhamnolipid synthesis by using the key enzymes and precursors from non-pathogenic sources is thought to not only eliminate pathogenic effects and simplify the downstream purification processes, but also to circumvent the complexity of quorum sensing system that regulates rhamnolipid biosynthesis. The pathogenicity of P. aeruginosa can also be reduced or eliminated through in vivo or in vitro enzymatic degradation of the toxins such as pyocyanin during rhamnolipid production. The rhamnolipid production cost can also be significantly reduced if rhamnolipid purification step can be bypassed, such as utilizing the fermentation broth or the rhamnolipid-producing strains directly in the industrial applications of rhamnolipids

Comparison of alkyl hydroperoxide reductase and two water-forming NADH oxidases from Bacillus cereus ATCC 14579

Bacillus cereus (B. cereus) is an ubiquitous facultative anaerobic bacterium, and its growth in aerobic environment correlates to the functions of its oxygen defense system. Water-forming NADH oxidase (nox-2) can catalyze the conversion of oxygen to water with concomitant NADH oxidation in anaerobic microorganisms. Here, we report the cloning and characterization of two annotated nox-2 s (nox-2(444) and nox-2(554)) from B. cereus ATCC 14579 and their comparison with another oxidative stress defense system alkyl hydroperoxide reductase (AhpR) from this microbe, which composed of two enzymes—hydrogen peroxide-forming NADH oxidase (nox-1) and peroxidase. Both nox-2 and AhpR catalyze the same reaction in the presence of oxygen. With the stimulation of exogenously added FAD, the maximum activity of nox-1, nox-2(444), and nox-2(554) could reach 27.7 U/mg, 22.9 U/mg, and 2.4 U/mg, respectively, at pH 7.0, 30 °C. Different from nox-1, both nox-2 s were thermotolerant enzymes and could maintain above 87% of their optimum activity at 80 °C, which was not found in other nox-2 s. As for operational stability, all are turnover-limited. Exogenously added reductive reagent dithiothreitol could dramatically increase the total turnover number of nox-2(444) and nox-2(554) by twofold and threefold, respectively, but had no effect on AhpR or nox-1

Random mutagenesis of global transcription factor cAMP receptor protein for improved osmotolerance

The naturally existing microbial hosts can rarely satisfy industrial requirements, thus there has always been an intense effort in strain engineering to meet the needs of these bioprocesses. Here, in this work, we want to prove the concept that engineering global transcription factor cAMP receptor protein (CRP) of Escherichia coli can improve cell phenotypes. CRP is one of the global regulatory proteins that can regulate the transcription of over 400 genes in E. coli. The target phenotype in this study is strain osmotolerance. Amino acid mutations were introduced to CRP by either error-prone PCR or DNA shuffling, and the random mutagenesis libraries were subjected to enrichment selection under NaCl stress. Five CRP mutants (MT1–MT5) were selected from the error-prone PCR libraries with enhanced osmotolerance. DNA shuffling technique was employed to generate mutant MT6 with MT1–MT5 as templates. All of these variants showed much better growth in the presence of NaCl compared to the wild type, and MT6 presented the best tolerance towards NaCl. In the presence of 0.9 M NaCl, the growth rate of MT6 is 0.113 h−1, while that of WT is 0.077 h−1. MT6 also exhibited resistance to other osmotic stressors, such as KCl, glucose, and sucrose. DNA microarray analysis showed that genes involved in colanic acid biosynthesis are up-regulated in the absence of salt stress, whereas carbohydrate metabolic genes are differentially expressed under NaCl stress when comparing MT6 to WT. Scanning electron microscopy images confirmed the elongation of both WT and MT6 when exposed to NaCl but the cell surface of MT6 was relatively smooth

Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance

One major challenge in biofuel production, including biobutanol production, is the low tolerance of the microbial host towards increasing biofuel concentration during fermentation. Here, we have demonstrated that Escherichia coli 1-butanol tolerance can be greatly enhanced through random mutagenesis of global transcription factor cyclic AMP receptor protein (CRP). Four mutants (MT1–MT4) with elevated 1-butanol tolerance were isolated from error-prone PCR libraries through an enrichment screening. A DNA shuffling library was then constructed using MT1–MT4 as templates and one mutant (MT5) that exhibited the best tolerance ability among all variants was selected. In the presence of 0.8 % (v/v, 6.5 g/l) 1-butanol, the growth rate of MT5 was found to be 0.28 h−1 while that of wild type was 0.20 h−1. When 1-butanol concentration increased to 1.2 % (9.7 g/l), the growth rate of MT5 (0.18 h−1) became twice that of the wild type (0.09 h−1). Microbial adhesion to hydrocarbon test showed that cell surface of MT5 was less hydrophobic and its cell length became significantly longer in the presence of 1-butanol, as observed by scanning electron microscopy. Quantitative real-time reverse transcription PCR analysis revealed that several CRP regulated, 1-butanol stress response related genes (rpoH, ompF, sodA, manX, male, and marA) demonstrated differential expression in MT5 in the presence or absence of 1-butanol. In conclusion, direct manipulation of the transcript profile through engineering global transcription factor CRP can provide a useful tool in strain engineering

Enhancing E. coli isobutanol tolerance through engineering its global transcription factor cAMP receptor protein (CRP)

The limited isobutanol tolerance of Escherichia coli is a major drawback during fermentative isobutanol production. Different from classical strain engineering approaches, this work was initiated to improve E. coli isobutanol tolerance from its transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP). Random mutagenesis libraries were generated by error-prone PCR of crp, and the libraries were subjected to isobutanol stress for selection. Variant IB2 (S179P, H199R) was isolated and exhibited much better growth (0.18 h−1) than the control (0.05 h−1) in 1.2% (v/v) isobutanol (9.6 g/L). Genome-wide DNA microarray analysis revealed that 58 and 308 genes in IB2 had differential expression (>2-fold, p < 0.05) in the absence and presence of 1% (v/v) isobutanol, respectively. When challenged with isobutanol, genes related to acid resistance (gadABCE, hdeABD), nitrate reduction (narUZYWV), flagella and fimbrial activity (lfhA, yehB, ycgR, fimCDF), and sulfate reduction and transportation (cysIJH, cysC, cysN) were the major functional groups that were up-regulated, whereas most of the down-regulated genes were enzyme (tnaA) and transporters (proVWX, manXYZ). As demonstrated by single-gene knockout experiments, gadX, nirB, rhaS, hdeB, and ybaS were found associated with strain isobutanol resistance. The intracellular reactive oxygen species (ROS) level in IB2 was only half of that of the control when facing stress, indicating that IB2 can withstand toxic isobutanol much better than the control. Biotechnol. Biotechnol

Improving ethanol tolerance of Escherichia coli by rewiring Its global regulator cAMP receptor protein (CRP)

A major challenge in bioethanol fermentation is the low tolerance of the microbial host towards the end product bioethanol. Here we report to improve the ethanol tolerance of E. coli from the transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP), which is known to regulate over 400 genes in E. coli. Three ethanol tolerant CRP mutants (E1– E3) were identified from error-prone PCR libraries. The best ethanol-tolerant strain E2 (M59T) had the growth rate of 0.08 h^-1 in 62 g/L ethanol, higher than that of the control at 0.06 h^-1. The M59T mutation was then integrated into the genome to create variant iE2. When exposed to 150 g/l ethanol, the survival of iE2 after 15 min was about 12%, while that of BW25113 was ,0.01%. Quantitative real-time reverse transcription PCR analysis (RT-PCR) on 444 CRP-regulated genes using OpenArrayH technology revealed that 203 genes were differentially expressed in iE2 in the absence of ethanol, whereas 92 displayed differential expression when facing ethanol stress. These genes belong to various functional groups, including central intermediary metabolism (aceE, acnA, sdhD, sucA), iron ion transport (entH, entD, fecA, fecB), and general stress response (osmY, rpoS). Six up-regulated and twelve down-regulated common genes were found in both iE2 and E2 under ethanol stress, whereas over one hundred common genes showed differential expression in the absence of ethanol. Based on the RT-PCR results, entA, marA or bhsA was knocked out in iE2 and the resulting strains became more sensitive towards ethanol.Published versio

Improving Acetate Tolerance of <i>Escherichia coli</i> by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)

<div><p>The presence of acetate exceeding 5 g/L is a major concern during <i>E. coli</i> fermentation due to its inhibitory effect on cell growth, thereby limiting high-density cell culture and recombinant protein production. Hence, engineered <i>E. coli</i> strains with enhanced acetate tolerance would be valuable for these bioprocesses. In this work, the acetate tolerance of <i>E. coli</i> was much improved by rewiring its global regulator cAMP receptor protein (CRP), which is reported to regulate 444 genes. Error-prone PCR method was employed to modify <i>crp</i> and the mutagenesis libraries (~3×10⁶) were subjected to M9 minimal medium supplemented with 5–10 g/L sodium acetate for selection. Mutant A2 (D138Y) was isolated and its growth rate in 15 g/L sodium acetate was found to be 0.083 h^-1, much higher than that of the control (0.016 h^-1). Real-time PCR analysis via OpenArray^® system revealed that over 400 CRP-regulated genes were differentially expressed in A2 with or without acetate stress, including those involved in the TCA cycle, phosphotransferase system, etc. Eight genes were chosen for overexpression and the overexpression of <i>uxaB</i> was found to lead to <i>E. coli</i> acetate sensitivity.</p> </div

Cell growth of <i>E. coli</i> DH5α overexpressing <i>uxaB</i> in M9 medium, with <i>E. coli</i> DH5α harbouring pCA24N as control.

<p>(<b>A</b>) 0 g/L sodium acetate (<b>B</b>) 5 g/L sodium acetate. Average values and standard deviations were calculated from triplicate experiments.</p

Growth profile.

<p>Mutant strain A2, the control (DH5α Δ<i>crp</i> harbouring plasmid pKCP containing native <i>crp</i> operon) and DH5α Δ<i>crp</i> strain harbouring blank plasmid pKC in M9 medium at 37 °C (<b>A</b>) 0 g/L sodium acetate (<b>B</b>) 10 g/L sodium acetate (<b>C</b>) 15 g/L sodium acetate. Average values and standard deviations were calculated from triplicate experiments.</p

Extracellular acetate concentration of A2 and the control.

<p>(<b>A</b>) M9 medium (0 g/L sodium acetate), normalized by cell OD600 values and (<b>B</b>) M9 minimal medium supplemented with 10 g/L sodium acetate. Average values and standard deviations were calculated from triplicate experiments.</p